Hybridomas and monoclonal antibodies that specifically bind to glutamic acid decarboxylase peptides

ABSTRACT

Isolated polypeptides useful in ameliorating GAD-associated autoimmune disease as well as diagnostic and therapeutic methods of using the peptides are disclosed. Monoclonal antibodies specific for GAD peptides and hybridoma cells producing such are also disclosed.

This is a divisional of application Ser. No. 08/123,859 filed Sep. 17,1993, now issued as U.S. Pat. No. 5,674,978, which is acontinuation-in-part of Ser. No. 07/716,909 filed Jun. 18, 1991, nowabandoned, which is a continuation-in-part of Ser. No. 07/586,536, filedSep. 21, 1990[, now abandoned].

BACKGROUND OF THE INVENTION

The present invention was supported by Grant NS22256 from the NationalInstitutes of Health. The United States Government has certain rights inthis invention.

1. Field of the Invention

The present invention relates to glutamic acid decarboxylase₆₅ (GAD₆₅)polypeptides and methods of using GAD₆₅ polypeptides diagnostically andtherapeutically in autoimmune disease.

2. Description of the Background Art

Insulin-dependent diabetes mellitus (IDDM; type I diabetes) is one ofthe most common metabolic disorders. In the United States, IDDM affectsapproximately one in 300 to 400 people, and epidemiological studiessuggest that its incidence is increasing. The disease results from theautoimmune destruction of the insulin-producing β-cells of the pancreas.More specifically, the preonset stage is characterized by "insulitis",in which lymphocytes infiltrate the pancreatic islets and selectivelydestroy the βcells. Insulitis may be present for many years before theonset of clinical symptoms. The typical IDDM presentation ofhyperglycemia appears only after at least 80% of the insulin-producingβ-cells are lost. The remaining β-cells are destroyed during the nextfew years.

Although insulin therapy allows most IDDM patients to lead normal lives,this replacement is imperfect and does not completely restore metabolichomeostasis. Thus, severe complications which result in dysfunctions ofthe eye, kidney, heart, and other organs are common in IDDM patientsundergoing insulin therapy. Because of this, it is highly desirable toextend the latency period and prevent progression (e.g., throughadministration of immunosuppressant drugs to interfere with theautoimmune process and insulin to achieve better control of the effectsof sustained hypoglycemia) between the start of β-cell destruction andthe actual requirement of insulin replacement (i.e., when 80% of theβ-cells are destroyed). Therefore, a diagnostic test which determinesthe beginning of β-cell destruction would allow the clinician toadminister immunosuppressant drugs (Silverstein, et al, New EnglandJournal of Medicine, 319:599-604, 1988) or prophylactic insulin therapy(Keller, et al., Lancet, 341:927, 1993) to extend this latency periodand thus significantly delay the onset of insulin replacement sideeffects.

Many IDDM patients have sera which contain antibodies to a 64 kDmolecule (Baekkeskov, et al., J. Clin. Invest., 79:926-934, 1987;Atkinson, et al., Lancet, 335:1357-1360,1990), to islet cell cytoplasmic(ICA) molecules or islet cell surface (ICSA) molecules (Bottazzo, etal., Lancet, 1:668-672, 1980), or to insulin (Palmer, et al., Science,222:1137-1139, 1983; Atkinson, et al., Diabetes, 35:894-898, 1986).Atkinson and coworkers (Atkinson, et al., Lancet, 335:1357-1360, 1990)have demonstrated that the presence of antibodies to the 64 kD moleculein human sera appears to be the earliest and most reliable indicatorthat onset of IDDM symptoms will eventually occur.

Recently, Baekkeskov and coworkers established that the 64 kD moleculeand glutamic acid decarboxylase (GAD) have several antigenic epitopes incommon and thus they may be identical or very similar molecules.Although this identification is an important finding, the use of thisinformation as a diagnostic tool for predicting IDDM is quite.cumbersome and limited unless knowledge of the molecular biology of GADis known. Studies by Kaufman, et al., (J. Clin. Invest., 89:283, 1992)established that the 64 kD molecule was intact GAD₆₅. Consequently, thecloning and subsequent production of large quantities of GAD₆₅ or a GADmolecule which is antigenically substantially identical to the GAD₆₅molecule or fragments of the GAD₆₅ molecule, both of which can be easilypurified, will allow the development of a diagnostic kit designed topredict IDDM as well as effective therapeutic modalities. The presentinvention provides a means for accomplishing these results.

SUMMARY OF THE INVENTION

The present invention arose out of the discovery that recombinant DNAtechnology could be used to produce eukaryotic GAD₆₅ polypeptide andthat GAD₆₅ polypeptide could be used in the diagnosis and therapy ofpatients with autoimmune disease. Particularly relevant is the use ofeukaryotic GAD₆₅ polypeptide in the diagnosis and therapy of patientshaving, or at risk of having, GAD-associated autoimmune disorders suchas insulin-dependent diabetes mellitus (IDDM) or stiff man disease.

A major advantage of the present invention is that it provides the artwith a ready source of eukaryotic GAD₆₅ polypeptide corresponding tothat purified from natural sources, while avoiding the problemsassociated with the isolation of naturally occurring eukaryotic GAD₆₅polypeptide when separating it from other eukaryotic non-GAD₆₅polypeptides. This absence of other eukaryotic non-GAD₆₅ polypeptides issignificant in that it allows the development of test systems which willonly detect antibodies specifically reactive with GAD₆₅ polypeptides.

Another advantage of providing eukaryotic GAD₆₅ polypeptide in hostcells is that by so doing, it is possible to obtain much largerquantities of the polypeptide than are currently practicably availablefrom natural sources. As a consequence, not only is it possible to usethe polypeptide of the invention to more accurately classify and treatpatients with such autoimmune diseases as IDDM, but it is also nowpossible to provide commercially useful quantities of GAD polypeptidefor use in diagnostic systems and pharmaceutical compositions.

DESCRIPTION OF THE DRAWINGS

FIG. 1 Cloning strategy for obtaining GAD₆₇ and GAD₆₇ specific cDNAprobes.

FIG. 2A, 2B and 2C DNA sequence and corresponding amino acid sequencefor rat GAD₆₅.

FIG. 3A, 3B, and 3D DNA sequence and corresponding amino acid sequencefor human GAD₆₅.

FIG. 4A and 4B Comparison of rat GAD₆₅ and human GAD₆₅ amino acidsequences.

FIG. 5 GAD₆₅ and GAD₆₇ cDNAs hybridize to different size RNAs.

FIG. 6 Southern blots hybridized with CDNA probes specific for GAD₆₅ andGAD₆₇.

FIG. 7 Immunological identification of GAD₆₅ and GAD₆₇.

FIG. 8 Proliferative T-cell responses of NOD mice to β cell antigens.

FIG. 9 Characterization of GAD specific T-cell response of NOD mice asprimed Th1 cells by enhanced clonal size (a) and cell surface markers(b) and IFNγ secretion.

FIG. 10 Intramolecular spreading of T-cell autoimmunity within the GADmolecule.

FIG. 11 Delay of onset of IDDM following immunization with GAD65.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the manipulation of genetic materialsby recombinant DNA procedures which make possible the production ofpolypeptides possessing part or all of the primary structuralconformation for one or more of the epitopes for binding autoantibodiesto glutamic acid decarboxylase₆₅ (GAD₆₅) and for polypeptides that bindto MHC receptors to block T-cell recognition. These polypeptides arehighly useful for the immunological detection of autoantibodies reactivewith them, since such autoantibodies are pre-diagnostic and indicativeof autoimmune diseases such as insulin dependent diabetes mellitus and"stiff man" syndrome. These polypeptides can also be used for purposesof screening drugs, such as those that alter GAD function, and forgeneration of polyclonal and monoclonal antibodies which, in turn, canbe used diagnostically to detect GAD₆₅.

The development of specific DNA sequences encoding eukaryotic GAD₆₅polypeptide for splicing into DNA vectors can be accomplished using avariety of techniques. For example, alternative methods which can beemployed include (1) the isolation of a double stranded DNA sequencefrom the genomic DNA of the eukaryote; (2) the chemical manufacture of aDNA sequence to provide the necessary codons for the polypeptide ofinterest; and (3) the in vitro synthesis of a double stranded DNAsequence by reverse transcription of mRNA isolated from a eukaryoticdonor cell. In the latter case, a double stranded DNA complement of MRNAis eventually formed which is generally referred to as CDNA.

The manufacture of DNA sequences is frequently the method of choice whenthe entire sequence of amino acid residues of the desired polypeptideproduct is known. When the entire sequence of amino acid residues of thedesired polypeptide is not known, the direct manufacture of DNAsequences is not possible and the method of choice is the formation ofcDNA sequences. Among the standard procedures for isolating cDNAsequences of interest is the formation of plasmid-carrying cDNAlibraries which are derived from reverse transcription of mRNA which isabundant in donor cells that have a high level of genetic expression.When used in combination with polymerase chain reaction technology, evenrare expression products can be cloned. In those cases where significantportions of the amino acid sequence of the polypeptide are known, theproduction of labeled single or double stranded DNA or RNA probesequences duplicating a sequence putatively present in the target cDNAmay be employed in DNA/DNA hybridization procedures which are carriedout on cloned copies of the cDNA which have been denatured into a singlestranded form (Jay, et al., Nucleic Acid Research, 11:2325, 1983).

Hybridization procedures are useful for the screening of recombinantclones by using labeled mixed synthetic oligonucleotide probes whereineach is potentially the complete complement of a specific DNA sequencein the hybridization sample which includes a heterogeneous mixture ofdenatured double stranded DNA. For such screening, hybridization ispreferably performed on either single stranded DNA or denatured doublestranded DNA. These procedures are particularly useful in the detectionof cDNA clones derived from sources where an extremely low amount ofmRNA sequences relating to the polypeptide of interest are present. Inother words, by using stringent hybridization conditions directed towardavoidance of non-specific binding, it is possible, for example, to allowthe autoradiographic visualization of a specific cDNA clone by thehybridization of the target DNA to that single probe in the mixturewhich is its complete complement (Wallace, et al., Nucleic AcidResearch, 9:879, 1981).

In addition, a GAD cDNA library can be screened by injecting the variouscDNAs into oocytes, allowing sufficient time for expression of the cDNAgene products to occur, and testing for the presence of the desired cDNAexpression product, for example, by using antibody specific for GAD₆₅polypeptide, by using functional assays for GAD₆₅ enzymatic activity, orby measuring the ability of the expression product to stimulatepathogenic T-cells.

Alternatively, a cDNA library can be screened indirectly for GAD₆₅peptides having at least one epitope using antibodies to GAD₆₅ (Changand Gottlieb, J. Neurosci., 8:2123, 1988). Such antibodies can be eitherpolyclonally or monoclonally derived and used to detect expressionproduct indicative of the presence of GAD₆₅ CDNA. Preferred areantibodies directed to an epitope found in the first 100 amino acids ofthe N-terminal portion of GAD₆₅.

Of the three above-noted methods for developing specific DNA sequencesfor use in recombinant procedures, the use of genomic DNA isolates, isthe least common. This is especially true when it is desirable to obtainthe microbial expression of mammalian polypeptides because of thepresence of introns.

The present invention provides novel polypeptides of GAD₆₅ which havepart or all of the primary structural conformation, that is, acontinuous sequence of amino acid residues, having at least one epitopefor antibodies to GAD₆₅ or at least one determinant for T-cellrecognition. It is possible to use the polypeptide fragments of theinvention rather than intact GAD to detect autoantibodies to GAD. Theterm "polypeptide," as applied to GAD polypeptide, includes any sequenceof amino acids having an epitope for autoantibodies to GAD or binds to aT-cell MHC receptor.

Thus, the polypeptide fragments of GAD encompassed by the inventionpossess a biological activity such as the ability to induce and/or bindautoantibodies to GAD, bind to T-cell MHC receptors (especiallyreceptors on pathogenic T-cells) and the like.

The polypeptides resulting from microbial expression of the DNAsequences of the invention or from other synthetic techniques, such assolid-phase peptide synthesis, can be further characterized by theirfreedom from association with other eukaryotic polypeptides or othercontaminants which might otherwise be associated with GAD in its naturalcellular environment or in such extracellular fluids as plasma or urine.

Studies by the present inventors unequivocally establish that GAD₆₅ andGAD₆₇ are encoded by distinct genes and are not produced, for example,by post-transcriptional or post-translational modification of a commongenomic sequence. Evidence proving that GAD₆₅ and GAD₆₇ are encoded bydifferent genes include: (a) the largest contiguous sequence of exactidentity between GAD₆₅ and GAD₆₇ cDNAs is only 17 nucleotides in length,(b) cDNAs from GAD₆₅ and GAD₆₇ do not cross hybridize with each other'sor with each other's mRNA under low stringency conditions (2.0×SSC,0.01% SDS, 23° C.), and (c) GAD₆₅ and GAD₆₇ cDNAs do not cross hybridizewith isolated genomic clones encoding GAD₆₇ and GAD₆₅, respectively.

The term "host" includes not only prokaryotes, but also such eukaryotesas yeast, filamentous fungi, plant and animal cells, as well as insectcells which can replicate and express an intron-free DNA sequence ofeukaryotic GAD₆₅. However, prokaryotes are preferred as the hostorganism for screening purposes while eukaryotic cells, especiallyinsect cells, are preferred for expression.

The term "prokaryotes" includes all bacteria which can be transformed ortransfected with the gene for the expression of GAD₆₅. Prokaryotic hostsmay include gram negative as well as gram positive bacteria such as, forexample, E. coli, S. typhimurium, Serratia marcescens and Bacillussubtilis.

A recombinant DNA molecule coding for the GAD₆₅ polypeptides can be usedto transform or transfect the host using any of the techniques commonlyknown to those of ordinary skill in the art. Especially preferred is theuse of a plasmid or a virus containing the GAD₆₅ coding sequence forpurposes of prokaryotic transformation or transfection, respectively.Alternatively, liposomes containing the DNA of interest can be used toobtain expression in the host (Zhu, et al., Science, 261:209, 1993)

Methods for preparing fused, operably linked genes and expressing themin bacteria are well-known in the art (Maniatis, et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., 1989). The genetic constructs and methods describedtherein can be utilized for expression of GAD₆₅ in prokaryotic hosts.

In general, expression vectors containing promotor sequences whichfacilitate the efficient transcription of the inserted eukaryoticgenetic sequence are used in connection with the host. The expressionvector typically contains an origin of replication, a promoter, and aterminator, as well as specific genes which are capable of providingphenotypic selection of the transformed cells. The transformedprokaryotic hosts can be grown in fermentors and cultured according totechniques known in the art to achieve optimal cell growth. Thepolypeptides of the invention can then be isolated from the grownmedium, cellular lysates, or cellular membrane fractions.

The isolation and purification of the expressed polypeptides of theinvention may be by any conventional means such as, for example,preparative chromatographic separations and immunological separationssuch as those involving the use of monoclonal or polyclonal antibody.

By having provided the sequence of amino acid residues of GAD₆₅, thepresent invention provides for the manufacture of DNA sequences whichcode for the host expression of polypeptide analogs or derivatives ofGAD₆₅ which differ from naturally-occurring forms in terms of theidentity or location of one or more amino acid residues and which sharesome or all of the epitopes of naturally-occurring polypeptide forms.

The novel DNA sequences of the invention include all sequences useful inproviding the expression in prokaryotic or eukaryotic host cells ofpolypeptides which have at least a part of the primary structuralconformation for one or more epitopes capable of reacting withautoantibodies to GAD₆₅ which are comprehended by: (a) the DNA sequenceas set forth in FIGS. 2 or 3 or their complementary strands; (b) DNAsequences which hybridize to DNA sequences defined in (a) or fragmentsthereof; and (c) DNA sequences which, but for the degeneracy of thegenetic code, would hybridize to DNA sequences defined in (a) and (b)above. Specifically comprehended in (b) are genomic DNA sequences whichencode allelic variant forms of GAD₆₅. Part (c) specifically comprehendsthe manufacture of DNA sequences which encode GAD₆₅, GAD₆₅ fragments,and GAD₆₅ analogs wherein the DNA sequences thereof may incorporatecodons which facilitate translation of mRNA in non-vertebrate hosts.

Since the cDNA sequence of the invention encodes essentially the entirehuman or rat GAD₆₅ molecule, it is now a matter of routine to prepare,subclone, and express smaller polypeptide fragments of cDNA from this ora corresponding cDNA sequence which would encode as few as one epitopefor autoantibodies to human or rat GAD₆₅. The presence of such anepitope on a cloned polypeptide can then be confirmed using, forexample, serum from a patient with autoantibodies to GAD₆₅. An exampleof such a smaller peptide is the first approximately 100 amino acidsfrom the N-terminus of GAD₆₅ (shown in FIG. 3). This amino acid sequenceis essentially absent from GAD₆₇. Other examples of specific peptides ofthe invention are shown in Table 7 as well as the approximatecarboxy-terminal two-thirds of GAD from about amino acid 224 to aboutamino acid 585. Especially preferred in the carboxy-terminal two-thirdsof GAD is the amino acid segment from about amino acid 224 to aboutamino acid 398.

The present invention further relates to monoclonal antibodies which arespecific for the polypeptides of the invention as well as the diagnosticand therapeutic use of these monoclonal antibodies. This specificityenables the monoclonal antibody, and like monoclonal antibodies withlike specificity, to be used to bind the polypeptide of the inventionwhen the polypeptide, or amino acids comprising the polypeptide, arepresent in specimens or a host, such as a human.

Numerous techniques can be utilized to produce the monoclonal antibodiesof the invention without resorting to undue experimentation. To a greatextent, the products of such monoclonal antibodies is rendered routinebecause of the highly defined nature of the polypeptides of theinvention. Thus, whether the polypeptides of the invention are used forimmunization and/or screening, the very limited number of immunogenicdeterminants on the polypeptides greatly simplifies the identificationof cell lines producing monoclonal antibodies of the invention, forexample, by limiting the repertoire of clonal expression possible.

One very useful type of cell line for expression of the monoclonalantibodies of the invention is the hybridoma. The general method usedfor production of hybridomas producing monoclonal antibody is well known(Kohler and Milstein, Nature, 256:495, 1975). The resulting hybridomaswere then screened for production of monoclonal antibodies capable ofbinding to the polypeptides of the invention.

The techniques of sensitization and/or immunization, cell fusion,ascites production, selection of mixed hybridomas, or subcloning ofmonoclonal hybridomas are generally well known in the art. Attention isdirected to Koprowski, et al., U.S. Pat. No. 4,172,124, Koprowski, etal., U.S. Pat. No. 4,196,265, or Douillard, J. Y. and Hoffman, T., BasicFacts about Hybridomas, in Compendium of Immunology, Vol. II, L.Schwartz, ed. (1981), which are herein incorporated by reference. Ingeneral, the purified peptides can be modified to have a cystineattached at the C-terminus to permit unidirectional attachment of thesynthetic peptide to an immunogenic protein through a connecting bridge,for example, maleimidobenzoylated (MB)-keyhole limpet hemocyanin (KLH).Other immunogenic conjugates can also be used, for example, albumin, andthe like. The resulting structure may have several peptide structureslinked to one molecule of protein.

Somatic cells derived from a host immunized against the syntheticpeptides can be obtained by any suitable immunization technique. Thehost subject is immunized by administering the antigen, usually in theform of a protein conjugate, as indicated above, by any suitable method,preferably by injection, either intraperitoneally, intravenously,subcutaneously, or by intra-foot pad. Adjuvants may be included in theimmunization protocol.

The initial immunization with the protein bound antigen can be followedby several booster injections given periodically at intervals of severalweeks. The antibody contained in the plasma of each host can then betested for its reactivity with the immunizing polypeptide of theinvention. The host having the highest response is usually mostdesirable as the donor of the antibody secreting somatic cells used inthe production of hybridomas. Alternatively, hyperimmunization can beeffected by repeatedly injecting additional amounts of peptide-proteinconjugate by intravenous and/or intraperitoneal route.

The isolation of hybridomas producing monoclonal antibodies of theinvention can be accomplished using routine screening techniques whichpermit determination of the elementary reaction pattern of themonoclonal antibody of interest. Thus, if a monoclonal antibody beingtested binds with a polypeptide of the invention, then the antibodybeing tested and the antibody produced by the hybridomas of theinvention are equivalent.

Alternatively, since the invention teaches polypeptides or amino acidsequences which are specifically required for binding of the preferredmonoclonal antibodies of the invention, it is now possible to use thesepeptides for purposes of immunization to produce hybridomas which, inturn, produce monoclonal antibodies specific for the polypeptide. Thisapproach has the added advantage of decreasing the repertoire ofmonoclonal antibodies generated by limiting the number of antigenicdeterminants presented at immunization, by the polypeptide. Themonoclonal antibodies produced by this method can be screened forspecificity using standard techniques, for example, by bindingpolypeptide to a microtiter plate and measuring binding of themonoclonal antibody by an ELISA assay.

It is also possible to determine, without undue experimentation, if amonoclonal antibody has the same specificity as a monoclonal antibody ofthe invention by ascertaining whether the former prevents the latterfrom binding the polypeptide of the invention. If the monoclonalantibody being tested competes with the monoclonal antibody of theinvention, as shown by a decrease in binding by the monoclonal antibodyof the invention, then it is likely that the two monoclonal antibodiesbind to the same, or a closely related, epitope.

Still another way to determine whether a monoclonal antibody has thespecificity of a monoclonal antibody of the invention is to pre-incubatethe monoclonal antibody of the invention with the polypeptide of theinvention with which it is normally reactive, and then add themonoclonal antibody being tested to determine if the monoclonal antibodybeing tested is inhibited in its ability to bind the antigen. If themonoclonal antibody being tested is inhibited then, in all likelihood,it has the same, or a closely related, epitopic specificity as themonoclonal antibody of the invention.

The GAD₆₅ of the invention is particularly suited for use inimmunoassays in which it can be utilized in liquid phase or bound to asolid phase carrier. In addition, GAD₆₅ used in these assays can bedetectably labeled in various ways.

Examples of immunoassays which can utilize the GAD₆₅ of the inventionare competitive and non-competitive immunoassays in either a direct orindirect format. Examples of such immunoassays are the radioimmunoassay(RIA), the sandwich (immunometric assay) and the Western blot assay.Detection of antibodies which bind to the GAD₆₅ of the invention can bedone utilizing immunoassays which run in either the forward, reverse, orsimultaneous modes, including immunohistochemical assays onphysiological samples. The concentration of GAD₆₅ which is used willvary depending on the type of immunoassay and nature of the detectablelabel which is used. However, regardless of the type of immunoassaywhich is used, the concentration of GAD₆₅ utilized can be readilydetermined by one of ordinary skill in the art using routineexperimentation.

The GAD and GAD fragments of the invention can be bound to manydifferent carriers and used to detect the presence of antibodyspecifically reactive with the polypeptide. Alternatively, thecarrier-bound GAD and GAD fragments can be used therapeutically forextracorporeal absorption of autoimmune antibodies in patients having,or at risk of having, GAD-associated disorders. Examples of well-knowncarriers include glass, polystyrene, polyvinyl chloride, polypropylene,polyethylene, polycarbonate, dextran, nylon, amyloses, natural andmodified celluloses, polyacrylamides, agaroses, and magnetite. Thenature of the carrier can be either soluble or insoluble for purposes ofthe invention. Those skilled in the art will know of other suitablecarriers for binding GAD₆₅, or will be able to ascertain such, usingroutine experimentation.

There are many different labels and methods of labeling known to thoseof ordinary skill in the art. Examples of the types of labels which canbe used in the present invention include enzymes, radioisotopes,colloidal metals, fluorescent compounds, chemiluminescent compounds, andbioluminescent compounds.

Alternatively, the polypeptide of the invention which comprises the GADenzymatic domain can be used to detect antibodies to GAD by measuringGAD enzymatic activity. For example, GAD₆₅. and a specimen suspected ofhaving antibodies to GAD₆₅ can be incubated for a period of time andunder conditions sufficient to allow binding to occur between GAD₆₅ andthe antibodies. The reaction product is precipitated and then tested forGAD enzymatic activity.

For purposes of the invention, the antibody which binds to GAD₆₅ of theinvention may be present in various biological fluids and tissues. Anysample containing a detectable amount of antibodies to GAD₆₅ can beused. Normally, a sample is a liquid such as urine, saliva,cerebrospinal fluid, blood, serum and the like, or a solid or semi-solidsuch as tissue, feces and the like.

The materials for use in the assay of the invention are ideally suitedfor the preparation of a kit. Such a kit may comprise a carrier meansbeing compartmentalized to receive in close confinement one or morecontainer means such as vials, tubes and the like, each of the containermeans comprising one of the separate elements to be used in the method.For example, one of the container means may comprise GAD₆₅ bound to acarrier. A second container may comprise soluble, detectably-labeledsecond antibody, in lyophilized form or in solution.

In addition, the carrier means may also contain a plurality ofcontainers each of which comprises different, predetermined amounts ofGAD₆₅. These latter containers can then be used to prepare a standardcurve into which can be interpolated the results obtained from thesample containing the unknown amount of autoantibodies to GAD₆₅.

In using the kit all the user has to do is add, to a container, apremeasured amount of a sample containing a measurable, yet unknownamount of autoantibodies to GAD₆₅ to be detected, a premeasured amountof carrier-bound GAD₆₅ present in the first container, and a premeasuredamount of the detectably labeled second antibody present in the secondcontainer. Alternatively, the non-detectably labeled GAD₆₅ can beprovided attached to the container to which the sample and thedetectably labeled second antibody are added. After an appropriate timefor incubation, an immune complex is formed and is separated from thesupernatant fluid, and the immune complex or the supernatant fluid aredetected, as by radioactive counting or addition of an enzyme substrate,and color development.

In an alternative embodiment, a kit comprising the GAD polypeptide ofthe invention can be used to detect the stage of GAD-associatedautoimmune disease in a patient. As further shown herein, Applicantshave discovered that certain GAD peptides or fragments are associatedwith different levels of progression in the autoimmune disease and thatthe level of disease process can be ascertained by looking at immunecell proliferative response, such as that of the pathogenic T-cell ofthe patient.

The term "ameliorate" denotes a lessening of the detrimental effect ofthe autoimmune response in the patient receiving therapy. The term"therapeutically effective" means that the amount of GAD₆₅ polypeptideused is of sufficient quantity to ameliorate the cause of disease due tothe autoimmune response.

The GAD₆₅ polypeptides, including whole GAD₆₅, of the invention can beused therapeutically in patients having, or at risk of having, anautoimmune response associated with GAD₆₅. Such therapy can beaccomplished, for example, by the administration of GAD₆₅ polypeptide toinduce tolerance to GAD. Such administration can utilize unlabeled aswell as labeled GAD₆₅ polypeptide. When unlabeled GAD₆₅ polypeptide isutilized advantageously, it would be in a form wherein, for example, theGAD₆₅ polypeptides are in fragments which are too small to stimulate animmune response, but large enough to bind, or block, the continuance ofthe autoimmune response. For example, GAD₆₅ could be digestedenzymatically into epitope-sized peptides (typically 5-12 amino acids inlength) and thereby bind to Fab binding portions present in the bodyfluids, or on the surface of immune cells, of the patient withautoimmune disease. Alternatively, peptides having at least onedeterminant for binding to T-cell MHC receptor can be similarly producedor chemically synthesized.

Alternatively, the GAD₆₅ polypeptides of the invention can beadministered labeled with a therapeutic agent. These agents can becoupled either directly or indirectly to the GAD₆₅ polypeptides of theinvention. One example of indirect coupling is by use of a spacermoiety. These spacer moieties, in turn, can be either insoluble orsoluble (Diener, et al., Science, 231:148, 1986) and can be selected toenable drug release from the GAD₆₅ polypeptide at the target site.Examples of therapeutic agents which can be coupled to the GAD₆₅polypeptides of the invention for immunotherapy are drugs,radioisotopes, lectins, and toxins.

The drugs with which can be conjugated to the GAD₆₅ polypeptides of theinvention include compounds which are classically referred to as drugssuch as mitomycin C, daunorubicin, and vinblastine.

In using radioisotopically conjugated GAD₆₅ polypeptides of theinvention for immunotherapy, certain isotopes may be more preferablethan others depending on such factors as leukocyte distribution as wellas stability and emission. Depending on the autoimmune response, someemitters may be preferable to others. In general, α and βparticle-emitting radioisotopes are preferred in immunotherapy.Preferred are short range, high energy α emitters such as ²¹² Bi.Examples of radioisotopes which can be bound to the GAD₆₅ polypeptidesof the invention for therapeutic purposes are ¹²⁵ I, ¹³¹ I, ⁹⁰ Y, ⁶⁷ Cu,²¹² Bi, ²¹¹ At, ²¹² Pb, ⁴⁷ Sc, ¹⁰⁹ Pd and ¹⁸⁸ Re.

Lectins are proteins, usually isolated from plant material, which bindto specific sugar moieties. Many lectins are also able to agglutinatecells and stimulate lymphocytes. However, ricin is a toxic lectin whichhas been used immunotherapeutically. This is accomplished by binding theα-peptide chain of ricin, which is responsible for toxicity, to theantibody molecule to enable site specific delivery of the toxic effect.

Toxins are poisonous substances produced by plants, animals, ormicroorganisms that, in sufficient dose, are often lethal. Diphtheriatoxin is a substance produced by Corynebacterium diphtheria which can beused therapeutically. This toxin consists of an α and β subunit whichunder proper conditions can be separated. The toxic A component can bebound to GAD₆₅ polypeptide and used for site specific delivery to aleukocyte expressing a receptor for GAD₆₅ polypeptide.

Other therapeutic agents which can be coupled to the GAD₆₅ polypeptidesof the invention, as well as ex vivo and in vivo therapeutic protocols,are known, or can be easily ascertained, by those of ordinary skill inthe art.

The present invention also relates to a polypeptide which can beadministered therapeutically to ameliorate, or utilized diagnosticallyto identify, the disease process in patients having, or at risk ofhaving, this disease. The conventional single-letter code used torepresent the various amino acids relates as follows:

                  TABLE 1                                                         ______________________________________                                        Phe: F      Leu: L     Ile: I     Met: M                                      Val: V      Ser: S     Pro: P     Thr: T                                      Ala: A      Tyr: Y     His: H     Gln: Q                                      Asn: N      Lys: K     Asp: D     Glu: E                                      Cys: C      Trp: W     Arg: R     Gly: G                                      ______________________________________                                    

A polypeptide sequence of the invention was identified by comparing theamino acid sequences of human GAD₆₅, human GAD₆₇, and the P2-C proteinof the picornavirus, coxsackie virus. The P2-C polynucleotide plays arole in the virus membrane bound replication complex. These analysesestablished the presence of an extensive sequence similarity betweenboth GAD₆₅ molecules and the coxsackie virus. A core polypeptide of sixcontiguous amino acid residues of the GAD₆₅ and P2-C polypeptide areidentical in amino acid sequence. Indeed, of the 24 amino acids in thepolypeptide, 19 are identical or conserved. In addition, there alsoexists a high charge density and the presence of a proline residue whichwould render this region highly antigenic (see Table 2).

                                      TABLE 2                                     __________________________________________________________________________    COMPARISON OF AMINO ACID SEQUENCES                                             Protein      Amino Acid Sequence                                             __________________________________________________________________________     ##STR1##                                                                     __________________________________________________________________________     The solid line encloses identical amino acids whereas the dashed line         encloses amino acid residues with similar charge, polarity, or                hydrophobicity.                                                          

In Table 2, the solid line encloses identical amino acids whereas thedashed line encloses amino acid residues with similar charge, polarity,or hydrophobicity.

The discovery of this common polypeptide region supports an etiologicrole for "molecular mimicry" in the precipitation of diabetes. Thus,where a patient genetically susceptible to IDDM is infected by acoxsackie virus, the immune response to the similar GAD sequence in thepatient's β-cells. The immunological response is maintained by theantigenically similar GAD polypeptides resulting in the eventualdestruction of the β-cells and the subsequent presentation of IDDM.

At present, it is believed that the destruction of pancreatic β-cells inIDDM is mediated by a cellular autoimmune response. As described herein,a polypeptide of the invention can ameliorate the autoimmune response toGAD. Because of the complexity of autoimmune disease, it is possible toenvision numerous possible therapeutic modalities which would allow thepolypeptides of the invention to be used to ameliorate such diseases. Inone embodiment, it appears that the polypeptides of the invention can beutilized to block recognition by a specific T cell receptor (TCR) or anMHC receptor presenting an autoimmune antigen on the surface of anantigen presenting cell (APC). The inhibition of such recognition mightoccur, for example, by providing the patient with the polypeptide of theinvention which, in turn, can displace the autoimmune antigen beingpresented in the antigen-cleft of the MHC receptor. However, althoughnot wanting to be bound to a particular theory, it is believed that thepolypeptides of the invention probably act to induce or restore atolerogenic state by direct interaction with the appropriate TCR on thesurface of a GAD specific pathogenic T-cell. This latter therapeuticapproach of direct interaction with the TCR is supported by the examplesand suggests that suppression of the autoimmune response can be achievedthrough induction of high-zone tolerance by use of high concentrationsof polypeptide, preferably soluble. Another possible mechanism is thatthe polypeptide of the invention may play a role in anergizingpathogenic T cells by binding to the T cell MHC receptor, therebypreventing the appropriate costimulatory signal.

Alternatively, the polypeptides of the invention could be used tostimulate a T-suppressor cell population in order to restoreself-recognition and, thereby, ameliorate the autoimmune disease.Stimulation of T-suppressor cell populations could be achieved, forexample, by use of a bi-specific antibody having one variable regionspecific for an epitope present on the autoimmune antigen residing inthe cleft of the MHCII receptor and, a second variable region specificfor an epitope present on the CD8⁺ receptor. The production of antibodyspecific for the polypeptide of the invention is a matter of routine tothose of skill in the art, as is the preparation of bi-specificantibodies having specificity for 2 or more epitopes.

Polypeptide analogs of the present invention may be designed which willcompete for recognition of self-antigens at the level of antigenpresentation or induce anergy in T cells, due to a lack of acostimulatory signal. Since MHC molecules contain a single peptidebinding site, it is possible to design polypeptides which will bind withhigh affinity to disease-associated MHC molecules, but will not activatedisease-causing T-helper cells. Such polypeptides act as antagonists forself-antigen recognition. In the present invention, support for thismechanism is found in the examples, especially Example 7. Precedent forsuch an approach arises from observation that a mouse lysozymepolypeptide, itself non-immunogenic, can compete for MHC binding with animmunogenic polypeptide from hen-egg white lysozyme and thereby reduce Tcell activation by that polypeptide (Adorini, et al., Nature,334:623-625, 1988) as well as studies using T-cell receptor peptides toblock formation of complex between T-cells, autoantigen and MHC (Howell,et al., Science, 246:668,1989). Similarly, such a therapeutic approachfor screening effective polypeptide analogs has been utilized in suchautoimmune diseases as experimental autoimmune encephalomyelitis (EAE)(Wraith, et al., Cell, 59:248, 1989; Urban, et al., Cell, 59:257, 1989).

The single-letter symbols used to represent the amino acid residues inthe polypeptides of the present invention are those symbols commonlyused in the art. The peptides of the invention include not only thenatural amino acid sequences, but also peptides which are analogs,chemical derivatives, or salts thereof. The term "analog" or"conservative variation" refers to any polypeptide having asubstantially identical amino acid sequence to a polypeptide providedherein and in which one or more amino acids have been substituted withchemically similar amino acids. For example, one polar amino acid, suchas glycine or serine, may be substituted for another polar amino acid;or one acidic amino acid, such as aspartic acid may be substituted foranother acidic amino acid, such as glutamic acid; or a basic amino acid,such as lysine, arginine, or histidine may be substituted for anotherbasic amino acid; or a non-polar amino acid such as alanine, leucine, orisoleucine may be substituted for another non-polar amino acid.

The term "analog" or "conservative variation" also means any polypeptidewhich has one or more amino acids deleted from or added to a polypeptideof the present invention, but which still retains a substantial aminoacid sequence homology to such peptide. A substantial sequence homologyis any homology greater than 70%, preferably at least about 80%, andmore preferably at least about 90%. The term "fragment" also means anyshorter version of the polypeptides identified herein having at least 6amino acid residues, wherein the fragment possesses biological activity,or is a fragment capable of inhibiting the stimulation of T-cells by astimulating polypeptide fragment or substantially full-length molecule.

The term "chemical derivative" means any polypeptide derived from apolypeptide of the present invention and in which one or more aminoacids have been chemically derivatized by reaction of the functionalside groups of amino acid residues present in the polypeptide. Thus, a"chemical derivative" is a polypeptide that is derived from thesequences or polypeptides identified herein by one or more chemicalsteps. Such derivatized molecules include, for example, those moleculesin which free amino groups have been derivatized to form aminehydrochlorides, P-toluene sulfoamides, benzoxycarboamides,T-butyloxycarboamides, thiourethane-type derivatives,trifluoroacetylamides, chloroxcetamides, or formamides. Free carboxylgroups may be derivatized to form salts, methyl and ethyl esters orother types of esters or hydrazides. Free hydroxyl groups may bederivatized to form O-acyl or O-alkyl derivatives. The imidazolenitrogen of histidine may be derivatized to form N-imbenzylhistidine.Also included as chemical derivatives are those polypeptides whichcontain one or more naturally occurring amino acids derivatives of the20 standard amino acids. For example, 4hydroxyproline may be substitutedfor proline; 5-hydroxylysine may be substituted for lysine;3methylhistidine may be substituted for histidine; homoserine may besubstituted for serine, and ornithine may be substituted for lysine.

It should be understood that the present invention is not limited to theillustrative polypeptides depicted in Table 2 and Table 9, instead, apolypeptide falling within the scope of this invention may extendoutside of, or comprise less than, the region between amino acid 28 andamino acid 50 of coxsackie virus P2-C, or between amino acid 250 andamino acid 273 of GAD₆₅, or between amino acid 258 and amino acid 281 ofGAD₆₇, as well as the region between amino acid 78 and amino acid 97, orbetween amino acid 247 and amino acid 266, or between amino acid 335 andamino acid 356, or between amino acid 479 and amino acid 498, or betweenamino acid 509 and amino acid 528, or between amino acid 524 and aminoacid 543, or between amino acid 539 and amino acid 556, or between aminoacid 564 and amino acid 583 of GAD₆₅, as long as a substantial part of agiven polypeptide is characterized by an amino acid sequence from thatregion, or segments or combinations thereof, and the polypeptidedemonstrates the desired immunological or biological activity againstautoimmune disease. In addition, polypeptides according to thisinvention include those having amino acid sequences which are longer orshorter in length than those of the polypeptides illustrated in Table 2and Table 9, or which comprise segments or combinations thereof, as longas such polypeptides consist substantially of the region between theamino acids illustrated in Table 2 and Table 9 and demonstrateimmunological or biological activity. All polypeptides of the inventionshould not stimulate or enhance the autoimmune disease.

Accordingly, it should be understood that the specific selection of anyone polypeptide within the polypeptides of the invention does notinvolve undue experimentation. Such a selection may be carried out bytaking a number of polypeptides and testing them for their immunologicaland biological activity in ameliorating the autoimmune disease or fordetecting antibody. The NOD mouse represents an excellent and wellcharacterized model for screening polypeptides of the invention capableof ameliorating or preventing diabetes. Example 7 illustrates anacceptable procedure for routine screening of candidate polypeptideswith biologic activity.

The polypeptides according to the present invention may be prepared byrecombinant techniques or by conventional synthesis using knownpolypeptide synthetic methods, including synthesis on a solid support.An example of a suitable solid phase synthetic technique is thatdescribed by Merriweather (J Am.Chem.Soc., 85:2149, 1963). Otherpolypeptide synthetic techniques may be found, for example, inBodanszky, et al., Peptide Synthesis, John Wiley & Sons, 2d ed., 1976,as well as other references known to those skilled in the art. A summaryof polypeptide synthesis techniques can be found in Stewart, et al.,Solid Phase Peptide Synthesis, Pierce Chemical Company, Inc., Rockford,Ill., 1984. The synthesis of polypeptides by solution methods may alsobe used, for example, as described in The Proteins, Vol. II, 3d ed.,Neurath, et al., eds., 105, Academic Press, New York, N.Y., 1976.Appropriate protective groups for use in such synthesis can be found inthe above references as well as in J. McOmie, Protective Groups inOrganic Chemistry, Plenum Press, New York, N.Y., 1973.

The polypeptides of the invention may also be prepared in an appropriatehost transformed with DNA sequences that code for the desiredpolypeptide. For example, a polypeptide may be prepared by thefermentation of appropriate hosts that have been transformed with andwhich express a DNA sequence encoding the polypeptide. Alternatively, aDNA sequence encoding several of the polypeptides of this invention maybe linked together and those sequences may then be used to transform anappropriate host to permit the expression of polypeptides involved inthe autoimmune disease.

The dosage ranges for the administration of the GAD polypeptides of theinvention are those large enough to produce the desired effect in whichthe symptoms or cellular destruction of the autoimmune response areameliorated. The dosage should not be so large as to cause adverse sideeffects, such as unwanted cross-reactions, anaphylactic reactions, andthe like. Generally, the dosage will vary with the age, condition, sex,and extent of the disease in the patient and can be determined by one ofskill in the art. The dosage can be adjusted by the individual physicianin the event of any counterindications. Dosage can vary from about 0.1mg/m² to about 2000 mg/m², preferably about 0.1 mg/m² to about 500 mg/m²/dose, in one or more dose administrations daily, for one or severaldays.

The GAD polypeptides of the invention can be administered parenterallyby injection or by gradual perfusion over time. The GAD polypeptides ofthe invention can be administered intravenously, intraperitoneally,intramuscularly, subcutaneously, intracavity, transdermally,intranasally, or enterally.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

The invention also relates to a method for preparing a medicament orpharmaceutical composition comprising the GAD₆₅ polypeptides of theinvention, the medicament being used for therapy of autoimmune responseto GAD₆₅.

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific examples which are provided herein for purposes of illustrationonly and are not intended to limit the scope of the invention.

EXAMPLE 1 CLONING AND EXPRESSION OF GAD₆₅

A. RECOMBINANT DNA PROCEDURES

In order to obtain cDNA probes specific for GAD₆₅ and GAD₆₇, total RNAwas extracted from adult rat brain by guanidine isothiocyanate-cesiumgradient using the method of Chirgwin, et al. (Biochemistry, 18:5294,1979). Poly (A) RNA was purified on oligo dT cellulose, using theprotocol by Bethesda Research Laboratories (BRL). First strand synthesiswas performed by using MMLV-reverse transcriptase (BRL), with conditionssuggested, except that poly d(N₆)-mers (Pharmacia) were used as primers.This cDNA-RNA mixture was heat inactivated at 65° C. for 15 min andstored at -20° C. For PCR, 1/50 of the sample was added to the 100 μlreaction. Degenerate oligonucleotides were synthesized (AppliedBiosystems) to encode the underlined common amino acid sequences offeline (from cDNA) (Kobayashi, et al., J. Neurosci., 7:2768, 1987) andrat (from peptides) (Chang and Gottlieb, J. Neurosci., 8:2123, 1988) GAD(FIG. 1). The 5'-end sequence of each degenerate oligonucleotidecontained one strand of the DNA sequence recognized by either Sstl andHindIII (5' oligo) or SstI and SstII (3'-end oligo). These primers wereused for selective amplification by polymerase chain reaction of thegenerated cDNA template as described by Gould, et al. (Proc. Natl. Acad.Sci.,USA, 86:1934, 1989). PCR products were subcloned into HindIII/SstIdouble digested Bluescript SK vector (Stratagene), transformed into DH5(BRL), and plated by standard methods (Maniatis, et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., 1989).

Colony hybridization was done with an 5'-³² P end labeledoligonucleotide specific to feline GAD₆₇ (Kobayashi, et al., J.Neurosci., 7:2768, 1987). End labeling of oligonucleotide, hybridizationconditions, and washing conditions were done as described (Wallace, etal., in Guide to Molecular Cloning Techniques; Berger, et al., Eds. inMethods of Enzymology; Abelson, et al., Eds. Academic Press, Inc., SanDiego, 432-442, 1987), except that the nitrocellulose filters werewashed at 50° C. for 15 min. Colonies which were positive and negativein the hybridization were individually picked and grown overnight inTerrific Broth (Tartof, et al., Focus, 9:12, 1987). DNA was isolatedusing a boiling method (Maniatis, et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., 1989) and templates were denatured by 0.2N NaOH and purified bySephacryl S400 spun columns (Pharmacia). Sequencing of denatured doublestranded template was by the chain-termination method (Sanger, et al.,Proc. Natl. Acad. Sci.,USA, 74:5463, 1977) using the T7-sequencing kit(Pharmacia).

As shown in FIG. 1, PCR-generated rat GAD₆₅ and GAD₆₇ cDNAs were used asprobes to screen a lambda ZAP (Stratagene) rat hippocampus libraryprovided by S. Heinemann (Salk Institute) by standard techniques(Maniatis, et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., 1989). A 2400 nucleotideGAD₆₅ cDNA (the largest clone) was isolated and subcloned by "zapping"as described by Stratagene. When a rat GAD₆₇ cDNA was obtained which wassmaller than a 3.2 kb rat GAD₆₇ cDNA clone already on hand, the largercDNA was sequenced. Exo III deletions (Henikoff, Gene, 28:351, 1984)were made in both directions for GAD₆₅ and GAD₆₇ and templates wereprepared and sequenced as described above. Anchored PCR (Frohman, etal., Proc. Natl. Acad. Sci.,USA, 85:8998, 1988) was done to clone theremaining 5'-ends of GAD₆₅ and GAD₆₇ mRNAs which were not represented inthe original cDNA clones isolated in the library screening. Sequencingof these clones revealed that neither GAD₆₅ nor GAD₆₇ mRNAs containedany further initiation codons (AUGs) in frame with the previouslydesignated initiation codons of the original cDNA clones.

EXAMPLE 2 CHARACTERIZATION OF CLONED GAD ₆₅

A. NORTHERN BLOT HYBRIDIZATION

Two PCR-derived cDNA probes were hybridized to Northern blots containingrat brain RNA in order to determine whether the GAD₆₇ and GAD₆₅ cDNAswere derived from two different mRNAs. RNA was extracted as described inExample 1. Poly (A) RNA was separated by electrophoresis in formaldehydeand transferred onto Biotrans (ICN) membranes, and hybridization wasperformed as described by Well, et al. (J. Neurosci., 16:311, 1986),except that 100 μl/ml of poly (A) was added. Probes were labeled toapproximately 10⁹ dpm/μg by the oligolabeling procedure of Feinberg andVogelstein (Anal. Biochem., 132:6, 1983). Identical results weresubsequently obtained with full-length clones of GAD₆₅ and GAD₆ cDNAs.

As shown in FIG. 5, lanes 1 and 2 contain 1 μg of poly (A) selected RNAextracted from rat cerebellum. Lane 1 was hybridized to a cDNA probe forthe rat cognate of feline GAD₆₇ (Kobayashi, et al., J. Neurosci.,7:2768, 1987) and lane 2 with a cDNA probe for the rat peptide sequence(which corresponds to GAD₆₅).

The cDNA probe for the rat peptide sequence hybridized to a 5.7 kb RNA,while the CDNA probe for the rat cognate of feline GAD₆₇ cDNA,hybridized to a 3.7 kb RNA. This demonstrates that GAD₆₅ and GAD₆₇ arenot derived from the same mRNA.

B. GENOMIC HYBRIDIZATION OF GAD₆₇ AND GAD₆₅

In order to investigate the possibility that GAD₆₇ and GAD₆₅ arise fromseparate genes, cDNAs of both GAD₆₇ and GAD₆₅ were hybridized to DNAblots containing genomic DNA.

For Southern blots, genomic DNA was extracted from rat liver asdescribed (Kaiser, et al., in DNA Cloning, vol.I, A Practical Approach,D. M. Glover ed., IRL Press, Oxford, pp. 38-40, 1985). DNA (10μg/sample) was digested to completion with EcoRI and HindIII usingconditions recommended by the suppliers (BRL, Gaithersburg, Md.). DNAfragments were separated by electrophoresis at 1.5 v/cm for 16 hrs in0.8% agarose. The DNA was then transferred to Zeta-Probe membranes(Bio-Rad), hybridized, and washed, as described by Gatti, et al.(Biotechniques, 2:148, 1984), except that 5 μg/ml Carnation dried milkwas substituted for Denhardt's solution. Probes for Southern blots werelabeled as described in Example 1, above.

As shown in FIG. 6, genomic DNA digested with HindIII and EcoRI are inlanes 1 and 3 and lanes 2 and 4, respectively. GAD₆₇ cDNA was hybridizedto lanes 1 and 2, whereas GAD₆₅ cDNA was hybridized to lanes 3 and 4.Numbers along the side of the gel are the DNA fragment sizes inkilobases.

This data shows that the two cDNAs hybridize to genomic fragments ofdifferent sizes. In addition, the greatest contiguous stretch ofidentical nucleotide sequence of GAD₆₅ and GAD₆₇ cDNAs is only 17nucleotide bases in length. Thus, GAD₆₇ and GAD₆₅ are encoded by twodistinct genes.

C. ENZYMATIC COMPARISON OF GAD₆₇ AND GAD₆₅

Studies were done comparing the effect of PLP on the activity of GAD₆₇and GAD₆₅. In so doing, both cDNAs were subcloned into vectors thatallowed their expression in bacteria (Studier, et al., J. Mol. Biol.,189:113, 1986). Overexpression of "fusionless" GAD₆₅ and GAD₆₇ wasaccomplished by subcloning GAD₆₅ cDNA into the NcoI site of pET-8c andGAD₆₇ cDNA into the NheI site of pET-5c vectors (Studier, et al., J.Mol. Biol., 189:113, 1986).

To obtain compatible sticky ends for correct in-frame subcloning of bothcDNAs, selective amplification was performed by PCR using conditionssuggested by United States Biochemical (USB), with 200 μM dNTPs and 1.5mM MgCl₂ in the mixture and annealing at 55° C. with 20 cycles todecrease infidelity of AmpliTAQ (USB). Primers specific for GAD₆₅ andGAD₆₇ contained one DNA strand of the NcoI and SpeI recognition sites,respectively. Since there is a NheI restriction site within the codingregion of GAD₆₇, SpeI (which is compatible with NheI) was used.

PCR products were subcloned into their respective pET vectors,transformed into DH5 and plated as described (Maniatis, et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y., 1989). Colonies were picked and grownovernight in LB broth with 50 μg/ml ampicillin. Subclones with correctorientation were transformed into BL21(DE3) strain (Studier, et al., J.Mol. Biol., 189:113, 1986) for overexpression. As a negative control,the pET-8C vector with no insert was transformed and subsequentlyinduced. Single colonies were picked, grown, induced by 1 mMisopropyl-B-D-thiogalacto-pyranoside (IPTG), and analyzed on SDS-PAGEgels as described (Sambrook, et al., Molecular Cloning a LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,17.15-17.16, 1989).

To measure GAD activity, we induced 10 ml cultures of bacteria at OD₆₀₀-0.5 with 1 mM IPTG. Two hours after induction, bacteria was spun downand resuspended and sonicated in 1 ml of homogenizing buffer (1 mMphenylmethylsulfonyl fluoride (PMSF), 1 mM 2-aminoethylisothiouroniumbromide (AET), and 60 mM potassium phosphate, pH 7.1). After sonication,cell debris was removed by centrifugation and protein concentration wasmeasured (Bradford, Anal. Biochem., 72:248, 1986) in the supernatant(supernatant was stored in aliquots at -70° C.). Brain homogenates wereprepared as described (Legay, et al., J. Neurochem., 46:1478, 1986). GADactivity was measured as described (Krieger, et al., J. Neurochem.,33:299, 1984) with 0.2 mM PLP present or absent and 20 μl of brainhomogenate or bacterial lysate in the incubation mixture. Production of¹⁴ CO₂ in bacterial lysates was linear with respect to time ofincubation and protein concentration.

                  TABLE 3                                                         ______________________________________                                                     GAD Specific Activity.sup.a                                                                Fold Increase                                       Source         - PLP     + PLP    in Induction                                ______________________________________                                        BL21(DE3) + pET-8c                                                                             12 ± 0.4                                                                            9 ± 1                                                                              --                                          BL21(DE3) + pET-GAD.sub.65                                                                   115 ± 3                                                                               773 ± 61                                                                           6.7                                         BL21(DE3) + pET-GAD.sub.67                                                                   160 ± 2                                                                              389 ± 8                                                                             2.4                                         Rat Brain      131 ± 5                                                                              216 ± 2                                                                             1.6                                         ______________________________________                                         .sup.a cpms of .sup.14 CO.sub.2 /μgprotein/hr of triplicates ±          S.E.M.                                                                   

As shown in Table 3, bacterial lysates containing GAD₆₅ or GAD₆₇catalyze the conversion of [1-¹⁴ C]-glutamate to GABA and ¹⁴ CO₂.

PLP stimulates the enzymatic activity of GAD₆₅ more than GAD₆₇. Thisgreater stimulation probably reflects the faster cycling of GAD₆₅through the inactivation cycle proposed by Martin and coworkers (Martin,Cell. Mol. Neurobiol., 7:237, 1987). This faster cycling suggests thatGAD₆₅ contributes more to the pool of apo-GAD that exists in vivo(Miller, et al., Brain Res. Bull., 5(Suppl.2):89, 1980). Thus, in vivo,PLP appears to regulate GAD₆₅ activity more than GAD₆₇ activity.

GAD₆₅ activity in bacterial lysates is similar to the five-fold PLPstimulation of GAD activity found in synaptosomes prepared from ratsubstantia nigra (Miller, et al., J. Neurochem., 33:533, 1979). Becauseboth GADs are more dependent upon added PLP in bacteria than is the GADactivity in crude rat brain homogenates, the endogenous PLPconcentration of bacteria lysates may be less than rat brainhomogenates.

D. IMMUNOLOGICAL IDENTIFICATION OF GAD₆₅ AND GAD₆₇

Rat brain homogenates and bacterial lysates were extracted as describedabove. Equal-volumes of loading buffer were added to each sample asdescribed (Harlow, et al., Antibodies, A Laboratory Manual, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., 1988). Proteins wereseparated by electrophoresis in a 10% acrylamide gel in SDS andelectrophoretically transferred to nitrocellulose (Harlow, et al.,Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y., 1988). The unreacted sites were blocked with aphosphate buffered saline (PBS) solution containing 2% bovine serumalbumin (fraction V), 1% gelatin, and 1% Triton-X-100 at 42° C. for onehr. After washing, the nitrocellulose filter was then cut into threesections and incubated with the following primary antibodies: lanes 1 to4 with a 1/2000 dilution of the antiserum of Oertel, et al.(Neuroscience, 6:2689, 1981), which recognizes both GAD₆₇ and GAD₆₅ ;lanes 5-8 with a 1/2000 dilution of K-2 antiserum, which recognizes onlyGAD₆₇ ; lanes 9-12 with a 1/2000 dilution of GAD-6 monoclonal antibody,which is specific for GAD₆₅ (Chang, et al., J. Neurosci., 8:2123, 1988).All filters were extensively washed and appropriate secondary antibodieswere incubated and washed. Bound antibodies were detected with ¹²⁵I-labeled protein A and autoradiography. Each lane contained thefollowing: lanes 1, 5, and 9 are BL21(DE3)+pET-GAD₆₇ ; lanes 2, 6, and10 are BL21(DE3)+pET-GAD₆₅ ; lanes 3, 7, and 11 are rat brainhomogenate; and lanes 4, 8, and 12 are BL21(DE3)+pET-8c.

The immunoblots of bacterially produced GAD₆₅ and GAD₆₇ demonstratedthat GAD₆₅ indeed corresponds to the smaller GAD in brain extracts, andGAD₆₇ to the larger form (FIG. 7). Previous work has demonstrated thecorrespondence of GAD₆₇ to the larger GAD for feline GAD₆₇, and formouse GAD₆₇ (Katarova, et al., Eur. J. Neurosci., 2:190, 1990; 235,1987). The mobilities of bacterially produced GAD₆₅ and GAD₆₇ (asdetected with the antiserum of Oertel, et al. (Neuroscience, 6:2689,1981) are identical to the immunoreactive doublet seen in rat brainhomogenate.

The smaller molecular weight and larger molecular weight forms of GAD inrat brain are thus identical in antigenicity and size to the products ofGAD₆₅ and GAD₆₇ cDNAs, respectively. Consequently, the two GADs in ratbrain are GAD₆₅ and GAD₆₇. From these data it can also be concluded thatthe molecular identity of the reported PLP-dependent and PLP-independentGADs by Tapia (Bayon, et al., J. Neurochem., 29:519,1977) are GAD₆₅ andGAD₆₇, respectively. Martin and coworkers (Spink, et al., Brain Res.,421:235, 1987) have reported the existence of four kinetically differentforms of rat brain GAD. However, immunoblotting experiments (with theantisera used here) of these forms have not been reported.

E. DISTRIBUTION OF GAD₆₅ and GAD₆₇ IN RNAs IN BRAIN TISSUE

Experiments were done to determine the distribution of GAD₆₅ and GAD₆₇in RNAs in cerebellum using in situ hybridization.

Transcripts of, respectively, 3.2 kb and 2.3 kb from GAD₆₅ and GAD₆₇cDNAs, were radiolabeled with ³⁵ S according to Wuenschell, et al.(Proc. Natl. Acad. Sci., USA, 83:6193,1986) procedure. Hydrolyzedfragments of 200 bp were hybridized to coronal sections of a ratcerebellum. Animals were anesthetized under halothane and decapitated.The brain was rapidly frozen in dry ice and coronal frozen sections (12μm) were fixed for 30 min in freshly prepared 4% formaldehyde inphosphate-buffered saline (PBS; 130 mM NaCl, 10 mM Na phosphate, pH7.0). The tissue was dehydrated through graded ethanol solutions andstored at -70° C.

In order to increase tissue permeability, the sections were submitted tothe following pretreatments: rehydration through graded ethanolsolutions (5 min each in 95%, 85%, 70%, 50%, and 30% ethanol); PBS (5min); 0.02N HCl (10 min); PBS (5 min); 0.01% Triton N-101 in PBS (1min); PBS (2×5 min); 1 μg/ml proteinase K (7.5 min); and glycine (toinhibit proteinase K) in PBS (3×5 min). Proteinase K was digested for 30min at 37° C. before use. Sections were then incubated at 37° C. in 50%formamide, 750 mM NaCl, 25 mM EDTA, 0.2% SDS, 0.02% BSA, 0.002% Ficoll,0.02% polyvinylpyrrolidone, 250 μg/ml yeast tRNA, 250 μg/ml poly A, and25 mM PPES (pH 6.8).

For the hybridization, 100 mM DTT, 10% dextran sulfate, and sense orantisense ³⁵ S-RNA were added to the prehybridization solution. Analiquot (50 μl) of the hybridization solution containing about 3 ng (10⁶cpm) of probe (sense or antisense) was added onto the slides. Each slidewas coverslipped and incubated for 16 hrs at 50° C., following which thesiliconized coverslips were removed by brief washing in 4×SSC (1×SSC-150mM NaCl, 60 mM Na citrate, pH 7.0).

Sections were then treated with ribonuclease A (50 μg/ml in 0.5M NaCl,10 mM Na thiosulfate, 1 mM EDTA, 10 mM TrisHCL, pH 8.0) for 20 min at37° C. and rinsed for 2 hrs at room temperature in 2×SSC, 10 mM Nathiosulfate, for 30 min at 55° C. Sections were dehydrated in ethanol,delipidated in xylene, coated with Kodak NTB2 emulsion and exposed for10 days at 4° C. The emulsion was developed with Kodak D19, and thetissue counterstained with cresyl violet.

Autoradiographic grains were detected using reflected polarized lightand grain numbers, densities, nd cell areas were determined with anAnalytic Imaging Concepts image analyzer system. Due to the lowbackground level, the criteria for defining a cell "labeled" was basedon the presence of more than 5 clustered grains. The GAD labeled cellswere found scattered throughout the brain, enabling the measurement ofthe number of grains over individual cells. The boundary of the cell andthe area covered by a grain allowed the calculation of the number ofgrains per cell. This analysis was done at a high magnification (800×),using both reflected polarized light and transmitted light tosimultaneously visualize the stained cell and the superimposed grains.Numbers are means ±S.E.M. of "n" cells.

                  TABLE 4                                                         ______________________________________                                               GRAINS/CELL                                                            CELL TYPE                                                                              GAD.sub.67 mRNA                                                                           GAD.sub.65 mRNA                                                                           GAD.sub.67 :GAD.sub.65                       ______________________________________                                        Purkinje 172 ± 34 (87).sup.a                                                                    43 ± 2 (70)                                                                            4.0                                          Golgi II 96 ± 8 (80)                                                                            64 ± 9 (65)                                                                            1.5                                          Basket    61 ± 12 (102)                                                                         16 ± 1 (57)                                                                            3.8                                          Stellate 55 ± 15 (65)                                                                           18 ± 3 (37)                                                                            3.1                                          ______________________________________                                         .sup.a ± S.E.M.(n)                                                    

In all neuronal types GAD₆₇ mRNA levels are greater. The observationswith in-situ hybridization are consistent with previous findings(Nitsch, J. Neurochem., 34:822, 1980; Denner, et al., J. Neurochem.,44:957, 1985; Itoh, et al., Neurochem. Res. 6:1283, 1981) that the ratioof PLP dependent to independent GAD activities in the cerebellum is oneof the lowest in brain regions tested. In addition, as shown in Table 3,the order of amounts for GAD₆₇ mRNA is Purkinje>Golgi II>Basket>Stellatecells; in contrast, for GAD₆₅ mRNA, this order is GolgiII>Purkinje>Basket>Stellate cells.

The expression of GAD₆₅. and GAD₆₇ mRNAs thus differs among classes ofneurons. The contribution of each to total GAD activity in turn affectshow GABA production is regulated. For example, the substantia nigracontains one of the highest ratios of PLP-dependent to PLP-independentGAD activities (Nitsch, J. Neurochem., 34:822, 1980). Increasing GABAconcentration in the substantia nigra by local injection of inhibitorsof GABA catabolism is especially effective in reducing seizuresusceptibility (Gale, Fed. Proc., 44:2414, 1985). Experimental animalsundergoing seizures induced by PLP-antagonists may therefore be unableto inhibit seizure propagation because of inhibition of GAD₆₅particularly in nerve terminals within the substantia nigra.

F. SUBCELLULAR LOCATION OF GAD₆₅ AND GAD₇

The distribution of GAD₆₅ and GAD₆₇ was evaluated in the S₂ andsynaptosome subcellular fractions. S₂ is a high speed supernatantconsisting of the cytosol of all cells in the brain, while thesynaptosomal fraction consists primarily of nerve endings (Gray, et al.,J. Anat., Lond, 96:79, 1962). For these studies, whole rat brainfractionation was performed as described by Booth and Clark (Booth, etal., Biochem. J., 176:365, 1978). Protein concentrations were determinedby Schaffner and Weissman (Schaffner, et al., Anal. Biochem. 56:502,1973). Samples were prepared as described (Kaiser, et al., DNA CloningVol. I, A Practical Approach, D. M. Glover ed. (IRL Press, Oxford, 1985,pp. 38-40), and immunoblotting was done as described above using GAD-6monoclonal antibody and K-2 antiserum. Equal amounts of protein (16 μg)were added to each lane . Autoradiography showed a linear response ofincreasing amount of ¹²⁵ I-protein A bound to antibody with proteinconcentrations of 1, 3, 10, 30, 100 μgs with both K-2 antiserum andGAD-6 monoclonal antibody (data not shown).

The results showed that GAD₆₇ was present in equal amounts in bothfractions. Since the n₂ fraction contains the cytosolic proteins ofglial (as well as other non-neuronal) and neuronal cells, theconcentration of GAD₆₇ must be greater in neuronal cell bodies than innerve endings. In contrast, the concentration of GAD₆₅ was greater insynaptosomes than in S₂, These subcellular fractionation experimentssuggest that, in contrast to GAD₆₅, a much greater fraction of GAD₆₇ ispresent in cell bodies of neurons than in nerve terminals. Thus,subcellular fractionation, like immunohistochemistry, shows that GAD₆₅and GAD₆₇ have different subcellular distributions.

In vivo experiments utilizing inhibitors of GABA synthesis anddegradation have suggested that the GABA pool in neuronal cell bodies isdifferent from that in the nerve terminals (ladarola, et al., Mol. Cell.Biochem., 39:305, 1981). GABA produced by GAD₆₇ may be involved more incellular metabolism (for example, in the GABA shunt) and indendrodendritic synapses. The dendrites of granule cells in theolfactory bulb, which form dendrodendritic synapses with mitraldendrites (Shepard, Physiol. Rev., 52:864, 1972) and probably releaseGABA (McLennan, Brain Res., 29:177-184,1971), label intensely with K-2antiserum. While not shown here, it has also been found greater GAD₆₇than GAD₆₅ mRNA levels (2-3 fold) in the olfactory bulb. Thisdistribution is consistent with the reported finding that most GADactivity in the olfactory bulb is present in S₂ and P₁ (crude nuclearpellet) and not in synaptosomes (Quinn, et al., J. Neurochem., 35:583,1980).

The differing subcellular distributions of GAD₆₅ and GAD₆₇ could resultfrom cytoskeletal anchoring or from some unknown protein targetingmechanism. Some cytoskeletal proteins have distributions that resembleGAD₆₅ and GAD₆₇. For example, in cultured sympathetic neurons Peng, etal. (J Cell. Biol., 102:252, 1986), demonstrate that 84% of tau is inaxons while 100% of MAP-2 is in cell bodies and dendrites. In addition,43 kd protein, a cytoskeletal protein, is thought to anchor theacetylcholine receptor to the underlying membrane cytoskeleton (Flucher,et al., Neuron, 3:163, 1989).

EXAMPLE 3 DETECTION OF GAD AUTOANTIBODIES IN CLINICAL SPECIMENS

A. MATERIALS AND METHODS

1. Patient Specimens. Sera from four groups of individuals were selectedfrom a previous study by Atkinson and co-workers (Atkinson, et al.,Lancet, 335:1357-1360, 1990). These groups consisted of: Group (1), 1new onset IDD patients diagnosed according to the established NationalDiabetes Data Group (NDDG) criteria (Gleichman, et al., Diabetes,36:578-584, 1987) that had been referred to the University of Florida,Diabetes Clinics; Group (2), 5 randomly selected islet cell cytoplasmicantibody (ICA) negative non-diabetic controls without any known familyhistory of autoimmune disease; Group (3), 13 individuals whose sera hadbeen collected 3 to 66 months prior to their documented clinical onsetsof IDD; Group (4), non-diabetic controls and relatives, and those whowere studied prior to their onsets of IDD; and Group (5), 3 patients atrisk for IDDM, but where onset has not yet occurred. This latter grouphad been ascertained through ongoing prospective ICA screening studiesof more than 5000 first degree relative of IDD probands, and 8200individuals from the general population (of which 4813 were schoolchildren).

2. Islet Cell Autoantibodies. ICA were assayed by indirectimmunofluorescence on blood group O cryocut pancreatic (Atkinson, etal., Lancet, 335:1357-1360, 1990). All results were interpreted on codedsamples, with control negative and positive sera in each batch. Thedegrees of ICA positivity were analyzed with the guidelines establishedby the Immunology Diabetes Workshop (IDW) for the standardization of ICA(Gleichman, et al., Diabetes, 36:578-584, 1987). All positive sera weretitered by end point dilution, and the Juvenile Diabetes Foundation(JDF) units were determined by reference to a standard serum previouslycalibrated to the international JDF standard of 80 units. In the studiesreported here, a positive ICA result was defined by replicate titers of10 JDF units or greater.

3. HLA DR Typing. HLA DR typing was performed as adapted from the methoddescribed by Van Rood and Van Leuwen (Nature, 262:795-797, 1976), usingDR trays (One Lamda Laboratories, Los Angeles, Calif.).

4. Human Islet Cells. Human pancreatic islets were isolated fromcadaveric pancreases and maintained in vitro as previously described(Ricordi, et al., Diabetes, 37:413-420, 1988). The islet cells weremetabolically labeled with ³⁵ S methionine (Amersham, Arlington Heights,Ill.) in vitro (95% air/5% CO₂).

5. Islet Cell Extractions and Immunoprecipitations. Islet cells wereextracted as previously described by Atkinson, et al. (Lancet,335:1357-1360, 1990) with the following modifications. Forimmunoprecipitation studies, the islet cell lysates were preclearedtwice by incubation (2 h, 4° C.) with either control, IDD serum (100μl), or GAD-6 (Chang, et al., J. Neuro, 8:2123-2130, 1988) (1 μl in 99μl of Tris buffer (Atkinson, et al., Lancet, 335:1357-1360, 1990) forevery 1000 islets. Immune complexes were then absorbed (1 h 4° C.) withan excess of protein A Sepharose CL-4B (Pharmacia, N.J.). Aliquotvolumes representing 1000 islet cells containing unbound (precleared)lysate were then incubated (12 h, 4° C.) with either IDD or control sera(25 μl), or GAD-6 (Chang, et al., J.Neuro, 8:2123-2130, 1988) (1 μl in25 μl Tris buffer). Following another incubation with protein ASepharose CL-4B (1 h, 4° C.), the complexes were then washed 5 timeswith 50 mM Tris HCL (pH 7.4) with 0.1% SDS, 1.0% Triton X-114, and 2 mMEDTA, and then washed again one time in double distilled water. Theprotein A Sepharose CL-4B was then boiled in Laemmli sample buffer(Laemmli, Nature, 227:680-685, 1970), and the samples were subjected toSDS-PAGE and fluororadiography (Kodak, X-omat AR5) using Enhance (NewEngland Nuclear). Alternatively, the autoradiographs were analyzed by aBETAGEN (Boston, Mass.) analyzer. Both 64 KA positive and negative serawere used in each assay, to serve as interassay controls. Allfluororadiographs were analyzed and rated as positive or negative aftercomparison with the known interassay controls. Positive serum sampleswere designated as 1 when a sample resulted in immunoprecipitation of alow intensity 64,000 M_(r) band, 2 if a moderate intensity band wasobserved and 3 if the intensity of the immunoprecipitated protein washigh. A similar rating procedure was employed for the intensity of bandscorresponding to immunoprecipitated ³⁵ S-GAD₆₅ and ³⁵ S-GAD₆₇.

6. Immunoprecipitations. Immunoprecipitation of bacterial lysatescontaining ³⁵ S-GAD₆₅ or ³⁵ S-GAD₆₇, and GAD from human brainhomogenate, was completed as described above in immunoprecipitationstudies of human islet cell extractions.

7. GAD Assays. Human brain homogenates were incubated with patient seraas described above in human islet cells. After absorption and washes,the protein A agarose slurry was aliquoted into three equal volumes andGAD activity was measured as described (Krieger, et al., Neurochem.33:299, 1984). Briefly, Protein A agarose beads were incubated with(1-¹⁴ C)-glutamate (Amersham) in a designated incubation mixture(Krieger, et al., J. Neurochem. 33:299, 1984) and production of ¹⁴ CO₂was quantitated by a liquid scintillation counter.

8. Production of ³⁵ S-GAD₆₅ and ³⁵ S-GAD₆₇. Rat GAD₆₅ and GAD₆₇ cDNAswere subcloned into a bacterial expression system as previouslydescribed. Labeling of ³⁵ S-GADs was completed by pulsing IPTG inducedbacterium (growing in Minimal Media) for 15 minutes with TRAN ³⁵ S-label(ICN). Cultures were then spun down and resuspended and sonicated in 1ml of homogenizing buffer (1 mM phenylmethylsulfonyl fluoride (PMSF), 1mM 2-aminoethylisothiouronium Bromide (AET) and 60 mM potassiumphosphate, pH 7.1). After sonication, cell debris was removed bycentrifugation and protein concentration was measured (Bradford, Anal.Biochem., 72:248, 1986) in the supernatant (supernatant was stored inaliquots at -70° C.).

B. IMMUNOREACTIVITY OF IDDM SPECIMENS

Sera from patients with IDDM were tested for the ability to precipitateGAD from human brain homogenates.

                  TABLE 5                                                         ______________________________________                                        SERA FROM                                                                     IDDM PATIENTS IMMUNOPRECIPITATE GAD ACTIVITY                                                 Pre-IDDM             GAD Activity.sup.4 4                      Patient                                                                             IDDM     Period.sup.1 1                                                                         64k.sup.22                                                                          JDF.sup.33                                                                          cpm's                                     ______________________________________                                        DA    .sup. *.sup.5                                                                          >24      3     164   13,762                                    DC    *        >1       3     20    1,719                                     RS    +         5       3     40    588                                       NL    +         0       2     80    440                                       DM    *        >1       2     10    184                                       C     -        na       0      0    280                                       C     -        na       0      0    285                                       C     -        na       0      0    325                                       C     -        na       0      0    275                                       C     -        na       0      0    270                                       ______________________________________                                         .sup.1 Expressed as months                                                    .sup.2 64K titers as described in Experimental Methods                        .sup.3 The islet cell antibody test as expressed in Juvenile Diabetes         Foundation (JDF) units                                                        .sup.4 Not adjusted for background                                            .sup.5 At risk for diabetes (also, failed glucose test)                       na -- Not applicable                                                     

As shown in Table 5, the sera of four (out of five) at risk for IDDM orIDDM patients bound significantly greater amounts of enzymaticallyactive GAD of human brain extracts than sera from control patients. Inaddition, sera from one of the patients was drawn in a pre-IDDM period,thus autoantibodies to GAD are present prior to the onset of IDDMsymptoms (see C below).

Further experiments (results not presented) showed that the sera of twoat risk IDDM patients (DA, DC) immunoprecipitated recombinantly produced³⁵ S-GAD₆₅ whereas recombinantly produced ³⁵ S-GAD₆₇ was only recognizedby sera of patient DA (and to a lesser degree than ³⁵ S-GAD₆₅).

Additional studies using patient DA sera showed the presence ofantibodies which recognize specific polypeptides produced in humanpancreatic islet cells. Electrophoretic analysis of the boundpolypeptides demonstrated the presence of autoantibodies to a 64 kDcomponent, as previously shown by others in human IDDM (Baekkeskov, etal., Nature, 298:167-169, 1982) and in animal models (Baekkeskov, etal., Science, 224:1348-1350, 1984; Atkinson, et al., Diabetes,37:1587-1590, 1988). Prior absorption of these sera with GAD-6monoclonal, which recognized GAD₆₅ but not GAD₆₇, or with bacteriallyproduced GAD₆₅, abolished the ability of the sera to recognize the 64 kDpancreatic polypeptide. The epitopes recognized by autoantibodies to the64 kD autoantigen are thus present in GAD₆₅, indicating that the 64 kDautoantigen is indeed GAD₆₅. In order to investigate the predictivevalue of GAD₆₅, sera drawn from patients prior to onset of clinicalmanifestation of IDDM were tested for autoantibodies to GAD₆₅.

                                      TABLE 6                                     __________________________________________________________________________    IDDM PATIENTS ANALYZED FOR                                                    AUTOANTIBODIES PRIOR TO THE ONSET OF DISEASE                                             Age Pre-IDD                                                        Patient                                                                           Sex HLA                                                                              Onset.sup.1 1                                                                     Period.sup.2 2                                                                      JDF                                                                              64KA.sup.3 3                                                                      GAD.sub.65.sup.33                                                                   GAD.sub.67.sup.33                           __________________________________________________________________________    TA  M   3,2                                                                              17  11    20 2   0     1                                           CA  F   4,5                                                                              38   4     0 1   1     0                                           RA  M   2,1                                                                               5  34     0 2   1     0                                           TB  M   2,4                                                                              11  66    40 1   1     0                                           AB  M   N.D.                                                                             23   6    160                                                                              3   3     2                                           VC  F   4,6                                                                              15   3    40 1   0     1                                           JD  M   6,1                                                                              34  25    10 3   1     1                                           DR  F   3,4                                                                              14  42    320                                                                              2   1     0                                           JG  M   3,3                                                                              12   8    40 1   0     0                                           BR  M   3,3                                                                               5   9     0 0   1     1                                           KR  F   4,x                                                                              34  14    10 3   2     0                                           JT  F   4,6                                                                               7  10    N.D.                                                                             1   1     1                                           __________________________________________________________________________     .sup.1 Age of IDDM onset expressed as months                                  .sup.2 The time interval between sera drawn and IDDM onset expressed as       months                                                                        .sup.3 1 = lowest; 2 = medium; and 3 = highest band intensities               N.D. -- not determined                                                   

As shown in Table 6, 9 out of 12 specimens (75%) were immunoreactivewith ³⁵ S-GAD₆₅. In addition, two patients (JA and VC) wereimmunoreactive to GAD₆₇, but not GAD₆₅ under these conditions.Therefore, in combination, autoantibodies to GAD₆₅ and GAD₆₇ werepresent in 11 out of 12 (91%) of these patients sera. This findingsuggests that although autoantibodies to GAD₆₅ are more common thanautoantibodies to GAD₆₇, the use of both recombinant GADs (GAD₆₅ andGAD₆₇) in an assay would allow for greater predictability of IDDM.Previous tests of these sera (Atkinson, et al., Langet, 335:1357-1360,1990) demonstrated that 11 out of 12, or 92%, immunoreacted With the ³⁵S-64 kD molecule from human pancreatic islet cells. The serum whichcontained detectable autoantibodies to the 64 kD molecule and not GAD₆₅was a serum which contained the lowest titer (or "1") for the 64 kDmolecule. Thus, the false negative obtained was due to a lack ofsensitivity in this assay. Furthermore, this assay predicted IDDM in onepatient (BR) who was negative for 64 K.

These results show that the 64 kD molecule identified in β-cells ofhuman pancreas is identical in size and antigenicity to rat GAD₆₅.Furthermore, sera drawn from patients prior to IDDM onset containautoantibodies to GAD₆₅. Consequently, the GAD₆₅ recombinant molecule isof great utility as a diagnostic tool for predicting IDDM. The abilityof a physician to diagnose IDDM prior to actual symptoms may result in agreater extension of time before insulin therapy is needed. Thesensitivity of such immunoassays will improve with the use of arecombinant GAD₆₅ of human origin which represents the GAD form presentin β-cells of the pancreas.

EXAMPLE 4 IMMUNE PROLIFERATIVE RESPONSE TO POLYPEPTIDE

Polypeptides were synthesized using an automatic instrument (AppliedBiosystems) and standard conditions. These polypeptides were then testedto compare their relative ability to stimulate proliferation of spleniclymphocytes and islet infiltrating T lymphocytes (IITLs). In this study,polypeptides derived from the GAD₆₅ core sequence and from thehomologous region of polio virus were compared. Appropriate cells werecultured for 5 days with the respective polypeptide in the presence of5×10⁴ irradiated spleen cells. ³ H-thymidine was added during the last16 hours of culture.

                                      TABLE 7                                     __________________________________________________________________________                            .sup.3 H-THYMIDINE INCORPORATION (cpm)                            AMINO ACID  BY LYMPHOID CELL POPULATION                           ANTIGEN     SEQUENCE    IITLs.sup.a                                                                            SPLEEN.sup.b                                 __________________________________________________________________________    None        --          1,100     6,500                                       Poliovirus SEQ ID NO:04                                                                   MKSMCPQAQLKVKYL                                                                             900    22,500                                       GAD.sub.65 SEQ ID NO:05                                                                   ARFKMFPEVKEKGMAA                                                                          9,500    23,300                                       __________________________________________________________________________     .sup.a islet infiltrating T lymphocytes (3 × 10.sup.4 cells/well)       .sup.b 1 × 10.sup.5 cells/well                                     

In these studies, there was no significant difference in theproliferative activity of cultures of spleen lymphocytes exposed toeither the polio or the GAD₆₅ polypeptides. However, both polypeptidesstimulated a T cell response which was higher than that found in themedia control. The lack of difference in proliferation in the spleencell population may be due to a lower frequency of GAD polypeptidespecific T cells.

The IITL population, when evaluated in the same manner, showed a markeddifference in cell proliferation. In this system, the response to theGAD₆₅ polypeptide was 9-fold greater than that of either the culturemedia or the polio polypeptide. This data strongly suggests that theGAD₆₅ is an important antigen for T cell responses in the IITLpopulation. This data suggests that molecular mimicry plays a role inthe pathogenesis of diabetes.

EXAMPLE 5 GAD INDUCES PROLIFERATION OF SPLEEN CELLS OF NOD MICE

Proliferative T-cell responses to β-cell antigens (RCA) developspontaneously in the nonobese diabetic (NOD) mouse model in a definedchronological order. The NOD mouse experimental model is considered themost analogous in vivo system available for studying IDDM in humans.This example describes studies on the antigen-induced blastogenesis ofspleen cells from newborn to 5 month old female NOD mice when exposed toGAD and other peptides.

The BCAs tested included one of the two forms of GAD (Kaufman, et al.,Science, 232:1138-1140, 1986; Erlander, et al., Neuron, 7:91-100, 1991;Kaufman, et al., Trends in Pharm. Sci. (in press)), (GAD₆₅, previouslyknown as the 64 K autoantigen (Baekkeskov, et al., Nature, 298: 167-169,1981; Baekkeskov, et al., Nature, 347:151-156, 1990), carboxypeptidase H(CPH) (Castano, et al., J. Clin. Endoctrinol. Metab., 73:1197-1201,1991), insulin (Palmer, Predicting IDDM, Diabetes Reviews, 1:104-115,1993) and a peptide of hsp which has been shown to be the immunodominantdeterminant recognized by NOD T-cells (Elias, Proc. Natl. Acad. Sci.,88:3088-3091, 1991). GAD in particular, is a good candidate for theinitial target antigen in IDDM since autoantibodies to GAD arise earlyin the natural history of the disease (Baekkeskov, supra; Atkinson, etal., Lancet, 335:1357-1360, 1990; Kaufman, et al., J. Clin. Invest.,89:283-292,1992). Furthermore, unlike the ubiquitous hsp, GAD isexpressed primarily in β-cells and the immunologically privilegedcentral nervous system (CNS) and gonads. As control antigens, irrelevantprototype foreign and self antigens including hen eggwhite lysozyme(HEL), human serum albumin (HSA), E. coli. β-galactosidase (β-gal) andmurine myelin basic protein (MBP) were used.

NOD (Taconic farms) and BALB/c mice (Jackson Laboratories) were keptunder specific pathogen free conditions. The mice were sacrificed at theages indicated and the spleen cells were tested directly ex vivo fortheir proliferative recall response to antigen. Single cell suspensionsof spleen cells were plated at 1×10⁶ cells per well in 96 wellmicrotiter plates in 200 μl serum free HL-1 medium (Ventrex) that wassupplemented with 2 mM glutamine with or without 10 μg/ml antigen (or 7μM peptide) in triplicate cultures. During the last 16 h of the 72 hculture period, 1 μCi[³ H]-thymidine was added per well. Incorporationof label was measured by liquid scintillation counting.

Both human GAD₆₅ (Bu, et al., Proc. Natl. Acad. Sci., 89:2115-2119,1992) and E. coli β-gal (control) were purified from recombinantbacteria on the basis of a hexahistidine tag which allows their rapidaffinity purification by metal affinity chromatography (Hochuli, et al.,Bio/Technology, 6:1321-1325, 1988). Bovine CPH was the generous gift ofL. Fricker (Albert Einstein Col. Med.) and human insulin was purchasedfrom Eli Lilly.

As illustrated in FIG. 8, while proliferative T-cell responses were notdetected at any time point to the control antigens, a response to GADarose at 4 weeks of age in NOD mice, concurrent with the onset ofinsulitis in the colony. The blastogenesis induced by GAD increasedduring the next four weeks and then declined to background levels byweek 16. At 6 weeks of age, near the peak of anti-GAD reactivity, T-cellresponses to hsp appeared and increased until week 15 and thendiminished as well (FIG. 8). In all NOD mice tested, hsp reactivity waspreceded by an anti-GAD response, suggesting that the former reactivitydeveloped as a secondary event during the autoimmune process. Similarly,while no response was detected to CPH at 4 weeks of age, a stronganti-CPH response was observed by week 8. In some mice, a weak responseto insulin was observed at 12 weeks, which became more prevalent at 15weeks of age (FIG. 8 and Table 8). None of the antigens inducedproliferation in T-cells from age-matched control BALB/c or (NOD×BALB/c)F₁ mice, both of which do not develop insulitis or IDDM. T-cellreactivity subsequently arises to other βCAs, consistent with theintermolecular diversification of the autoimmune response. Thus, theautoimmune response to GAD was the first to occur among the autoantigenstested. In view of this, tolerization to GAD should prevent the spreadof autoimmunity to other βCAs and insulitis. If this were not the casethen tolerization to GAD should have no effect on the response to theseother antigens.

Blastogenesis provides an approximation of the relative clonal sizes ofantigen-specific CD4+T-cells (Corradin, et al., J. Immunol., 119:1048-1053, 1977). The data in FIG. 8 shows that GAD reactive T-cells"spontaneously" undergo clonal expansion concurrent with the onset ofinsulitis. These findings are consistent with an endogenous primingevent.

EXAMPLE 6 INDUCTION OF TOLERANCE WITH GAD

This example describes a study which shows that induced tolerance to GADcan ameliorate IDDM.

1. In these experiments female NOD mice were intravenously injected at 3weeks of age with 50 μg GAD, β-galactosidase, mycobacterial hsp65(m-hsp) or 0.1 μg of the immunodominant hsp peptide (hsp-p), in PBS. At12 weeks of age, mice were examined for insulitis and autoantigenreactive T-cells. At this age both indications are established inuntreated NOD mice. Pancreatic tissue sections were stained byimmunoperoxidase techniques for insulin and were counterstained withhematoxylin. Insulitis was scored in a blinded manner by examining 54 to87 islets on 5 interrupted tissue sections from each pancreas.Proliferative splenic T-cell responses induced by various antigens wereperformed as described above in Example 4. Data in Table 8 are expressedas the average [³ H]thymidine label (cpm) incorporated in triplicatecultures.

                                      TABLE 8A                                    __________________________________________________________________________    GAD Induced Tolerance                                                                     Spleen Cell Proliferation (SI ± SEM).sup.b                     Insulitis               GAD Peptides    hsp                                   Treatment                                                                          Score.sup.a                                                                        N β-Gal                                                                          GAD   #17   #34  #35  Peptide                                                                             CPH                             __________________________________________________________________________    Uninjected                                                                         2.4 ± 0.2                                                                       5 1.0 ± 0.2                                                                        9.5 ± 2.1                                                                        4.8 ± 0.4                                                                        6.0 ± 0.1                                                                       2.9 ± 0.2                                                                       6.7 ± 1.0                                                                        ND.sup.c                        β-Gal.                                                                        2.6 ± 0.6                                                                       5 1.1 ± 0.1                                                                        15.4 ± 1.8                                                                       5.1 ± 0.6                                                                        5.1 ± 0.6                                                                       4.0 ± 0.2                                                                       6.6 ± 0.5                                                                        11.5 ± 0.9                   GAD  0.1 ± 0.1                                                                       8  1.1 ± 0.03                                                                      1.6 ± 0.3                                                                         1.0 ± 0.05                                                                      1.2 ± 0.1                                                                       1.0 ± 0.1                                                                       1.2 ± 0.1                                                                         1.1 ± 0.02                  hsp-p                                                                              1.7 ± 0.4                                                                       5  1.1 ± 0.05                                                                      5.8 ± 0.2                                                                        4.5 ± 0.1                                                                        4.1 ± 0.3                                                                       4.2 ± 0.1                                                                        1.1 ± 0.04                                                                      4.4 ± 0.2                    m-hsp                                                                              1.8 ± 0.5                                                                       5 1.0 ± 0.1                                                                        4.2 ± 0.1                                                                        3.9 ± 0.1                                                                        3.9 ± 0.1                                                                       3.4 ± 0.2                                                                        1.0 ± 0.03                                                                      4.3 ± 0.2                    __________________________________________________________________________     .sup.a Severity of mononuclear cell infiltration was defined                  histologically (0 = no lymphocytic infiltration; 1 = <25%; 2 = 25-50%; 3      50-75%; 4 = >75%)(Qln, et al., Immunol., 150: 2072-2080, 1993). Score is      mean ± SE.                                                                 .sup.b Significant responses noted by solid underline, borderline             responses noted by double underline.                                          .sup.c Not determined.                                                   

                                      TABLE 8B                                    __________________________________________________________________________    GAD Induced Tolerance                                                                     Spleen Cell Proliferation (SI ± SEM).sup.b                     Insulitis              GAD Peptides      hsp                                  Treatment                                                                          Score.sup.a                                                                        N β-Gal                                                                         GAD   #17   #34   #35   Peptide                                                                             CPH                            __________________________________________________________________________    11 peptide                                                                         ≈2.5                                                                         1.0 ± 0.1                                                                       21.1 ± 2.2                                                                       13.7 ± 1.5                                                                       11.4 ± 1.5                                                                       11.3 ± 0.7                                                                       13.3 ± 0.9                                                                       ND                             34/35                                                                              0.7 ± 0.4                                                                         1.0 ± 0.2                                                                       1.9 ± 1.1                                                                        2.2 ± 1.2                                                                        1.1 ± 0.3                                                                        1.0 ± 0.1                                                                        1.8 ± 1.1                                                                        ND                             peptides                                                                      (+IFA)                                                                        IFA alone                                                                          ≈2.5                                                                         1.0 ± 0.1                                                                       8.1 ± 0.5                                                                        5.0 ± 0.5                                                                        4.8 ± 0.5                                                                        4.8 ± 0.4                                                                        6.6 ± 0.6                                                                        ND                             (+IFA)                                                                        __________________________________________________________________________     .sup.a Severity of mononuclear cell infiltration was defined                  histologically (0 = no lymphocytic infiltration; 1 = <25%; 2 = 25-50%; 3      50-75%; 4 = >75%)(Qln, et al., Immunol., 150: 2072-2080, 1993). Score is      mean ± SE.                                                                 .sup.b Significant responses noted by solid underline, borderline             responses noted by double underline.                                          .sup.c Not determined.                                                   

                                      TABLE 8C                                    __________________________________________________________________________    GAD Induced Tolerance                                                                      Spleen Cell Proliferation (SI ± SEM).sup.b                    Insulitis               GAD Peptides   hsp                                    Treatment                                                                           Score.sup.a                                                                        N β-Gal                                                                         GAD   #17  #34  #35  Peptide                                                                            CPH                               __________________________________________________________________________    11 peptide                                                                          ≈(?)                                                                         1.4 ± 0.4                                                                       12.1 ± 3.5                                                                       6.9 ± 0.4                                                                       6.4 ± 1.0                                                                       6.8 ± 0.3                                                                       8.7 ± 0.9                                                                       ND                                HEL peptide                                                                         (?)    1.2 ± 0.2                                                                       10.7 ± 3.5                                                                       6.4 ± 1.4                                                                       5.9 ± 1.6                                                                       6.9 ± 1.9                                                                       8.0 ± 1.8                                                                       ND                                34/35 (?)    1.0 ± 0.1                                                                        4.4 ± 2.6                                                                       1.8 ± 1.1                                                                       1.0 ± 0.1                                                                       1.1 ± 0.2                                                                       2.7 ± 1.3                                                                       ND                                peptides                                                                      (+IFA)                                                                        __________________________________________________________________________     .sup.a Severity of mononuclear cell infiltration was defined                  histologically (0 = no lymphocytic infiltration; 1 = <25%; 2 = 25-50%; 3      50-75%; 4 = >75%)(Qln, et al., Immunol., 150: 2072-2080, 1993). Score is      mean ± SE.                                                                 .sup.b Significant responses noted by solid underline, borderline             responses noted by double underline.                                          .sup.c Not determined.                                                   

Seventy five percent of the GAD treated mice, but none of the controls,showed no T-cell reactivity to GAD (indicating complete tolerization) orto other βCAs. These mice were also completely free of insulitis (score0.0). If there were another effector T cell population in the islets,specific for an unknown βCA, that preceded the anti-GAD response, therelease of cytokines by this population should have promoted T-cellresponses to βCAs and insulitis (Sarvetnick, et al., Nature, 346:844,1990; Heath, et a/., Nature, 359:547, 1992). Twenty five percent of theGAD-treated mice were not completely tolerized to GAD, as evidenced by aweak residual GAD reactivity (SI of about 3) and displayed very limitedperi-insulitis. In contrast, while tolerization to both of the hspantigens was complete, these treatments reduced, but did not prevent,the development of T cell responses to other βCAs or insulitis. Thus,while the inactivation of GAD-reactive T cells prevented β cellautoimmunity, hsp tolerization only partially reduced it, as would beexpected if a secondary element was removed from the amplifactorycascade.

In ongoing experiments examining the effects of GAD tolerization ondiabetes incidence, all of the GAD treated mice (n=17, presently 37weeks old) have normal glucose levels, while 70% of the mice receivingcontrol antigens developed hyperglycemia by 19 weeks of age (n=20). FiveGAD treated mice were sacrificed at 30 weeks of age. All were free ofdetectable βCA reactive T cells. Of these five animals, four mice werecompletely free of insulitis and one mouse displayed very limitedperi-insulitis. These data show that inactivation of GAD reactiveT-cells prevents the long term development of insulitis and diabetes.

2. In a second set of experiments neonatal female NOD mice were injectedintraperitoneally IFA with peptide 11, a mixture of peptides 34 and 35plus IFA, or with IFA alone and at 12 weeks of age the mice wereexamined for insulitis and autoantigen reactive T-cells as in Example6.1. Proliferative splenic T-cell responses induced by the variousantigens were performed as in Example 4, and data in Table 8B areexpressed as the average [³ H]-thymidine label (cpm) incorporated intriplicate cultures.

The data in Table 8B show that tolerization with control peptide 11 didnot prevent auto antibody response to GAD or to GAD peptides 17, 34 or35. Nor was response to hsp peptide prevented by tolerization withpeptide 11. IFA alone was somewhat effective at suppressing immuneresponse. By contrast, tolerization with the mixture of peptides 34 and35 suppressed the autoimmune response of spleen cell proliferation toall species tested: β-gal, GAD, GAD peptides 17, 34 and 35, and hsppeptide. In addition, tolerization to GAD peptides 34 and 35 greatlyreduces insulitis but does not completely prevent it as whole GAD₆₅does.

3. In a third set of experiments female NOD mice were intravenouslyinjected at three weeks of age with peptide 11 (control), a mixture ofpeptides 34 and 35 plus IFA, or with HEL peptides and at 12 weeks of agethe mice were examined for insulitis and autoantigen reactive T-cells asin Example 6.1. Proliferative splenic T-cell responses induced by thevarious antigens were performed as in Example 4, and data in Table 8Care expressed as the average [³ H]-thymidine label (cpm) incorporated intriplicate cultures.

The data in Table 8C show that tolerization with control peptide 11 didnot prevent auto antibody response to GAD, to GAD peptides 17, 34 or 35or to hsp although the response was not as great as in Example 6.2. Norwas response to hsp peptide prevented by immunization with peptide 11.By contrast, immunization with the mixture of peptides 34 and 35 plusIFA suppressed the autoimmune response of spleen cell proliferation toall species tested: β-gal, GAD, GAD peptides 17, 34 and 35, and hsppeptide.

EXAMPLE 7 CHARACTERIZATION OF GAD-REACTIVE T-CELLS

This example describes studies on GAD-Reactive T-Cells for additionalproperties that distinguish activated/memory from resting/naivelymphocytes.

In one series of experiments γ interferon (IFNγ) was measured by ELISAin culture supernatants (CSN) of spleen cells of 6-9 week old mice afterchallenge with GAD or control antigens HEL and MBP. Additionally, thefrequency of antigen specific, IFNγ-producing cells was determined by anELISA spot technique (T. Taguchi, et al., J. Immunol., 145:68-77, 1990).Frequency of antigen-induced, spot forming cells (SFC) among 10³ spleencells is represented in FIG. 9(a). Values are the mean+SEM from 5individual female NOD mice, each tested in triplicate cultures with orwithout antigen. Results from a single experiment are shown. These arerepresentative of 3 separate experiments.

In performing these experiments, freshly isolated spleen cells werecultured with or without antigen as described in Example 4. CSN weretaken after 48 h and the concentration of IFNγ was determined by ELISA(Macy, et al., FASEB J., 3003-3009,1988). IFNγ specific monoclonalantibody (mAb) R4-6A2 (Pharmingen) was used as the capturing reagent andbiotinylated mAb XMG 1.2 (Pharmingen, also specific for IFNγ) was usedin conjunction with streptavidin-alkaline phosphatase (Zymed) andp-nitrophenol for detection of bound lymphokine. Recombinant murine IFNγ(Pharmingen) was used as a standard. ELISA spot assays for the detectionof antigen-specific, IFNγ-producing cells were performed as described(Taguchi, et al., J. Immunol., 145:68-77, 1990). After a 24 hpre-activation culture of spleen cells with our without antigen, cellswere transferred by serial dilution to 96 well microtiter plates(Millipore) that had been pre-coated with mAb R4-6A2. After 24 h, thecells were removed and IFNγ spots were visualized using XMG 1.2-biotinin conjunction with nitroblue terazolium-bromochloroindolyl phosphatesubstrate (Sigma). Spots were counted visually and the frequency ofantigen specific cells was determined from the difference between thenumber of spots seen with and without antigen.

As shown in FIG. 9(a), when freshly isolated T-cells from 6-9 week oldNOD mice were challenged with GAD or control antigens, highconcentrations of IFNγ were detected only in cultures containing GAD,suggesting that the GAD specific T-cells had been pre-activated in vivo,since only pre-activated T-cells (Th1) produce IFNγ within 48 hoursafter antigen recognition (Ehlers, et al., J. Exp. Med., 173:25-36 1991;Croft, et al., J. Exp. Med., 176:1431-1437, 1992). In contrast, T-cellsfrom age matched BALB/c mice did not respond to GAD or to controlantigens by IFNγ production (data not shown).

Results of the ELISA spot assay to measure directly the frequency ofGAD-specific T-cells showed that while in 6-9 week old NOD mice, T-cellsreactive to control antigens constituted approximately 1 in 10⁵ cells inthe spleen, the frequency of GAD-reactive T-cells was about two ordersof magnitude higher, ranging from 90-291 cells per 10⁵ cells (FIG. 9(a),confirming the data obtained by proliferation assays (FIG. 8) that thesecells had been clonally expanded in vivo.

In another series of experiments, GAD specific T-cells werecharacterized for expression of the cell surface marker L-selectin,since murine T-cells convert from an L-selectin⁺ (L-sel⁺) to anL-selectin⁻ (L-sel⁻) phenotype upon activation (Bradley, et al., J.Immunol., 148:324-331, 1992).

To perform these studies, pooled spleen cells from 3 to 4 age matchedmice were panned on plates coated with goat-anti-mouse Ig (Zymed) toremove adherent macrophages as well as B cells. Next, CD8+ cells werecoated with mAb 58.6-72 (ATCC) and removed by panning over plates coatedwith goat-anti-rat Ig (Zymed). The non-adherent CD4+ cell fraction waslabeled with anti-L-selectin mAb MEL-14 (ATCC) and panned ongoat-anti-rat Ig coated plates. Both the adherent (CD4+L-sel⁺) andnon-adherent (CD4+,L-sel⁻) fractions were sampled. Purity of the cellfraction was assessed by FACS analysis; cells were >90% CD4+ and >95%enriched for the L-sel⁻ or L-sel⁺ phenotype. The purified cell fractionswere tested for GAD reactivity by seeding them at 2×10⁵ cells per wellin 96 well microtiter plates with or without antigen. Irradiated (3000rad), unseparated spleen cells of 3 week old NOD mice were added at5×10⁵ cells per well as a source of antigen presenting cells.Supernatants of triplicate cultures were taken 48 h later and their IFNγcontent was determined by ELISA.

The results of this study showed that by 2-3 weeks of age, GAD reactiveT-cells could not be detected in either the L-sel⁺ or the L-sel⁻population, consistent with a low frequency of antigen reactiveprecursors at this time point. However, by 6 weeks of age high levels ofIFNγ were induced by GAD (but not by control antigens) in the L-sel⁻(but not the L-sel⁺) subpopulation of CD4+ cells (FIG. 9(b)).

The increase in clonal size of GAD reactive T-cells, their production ofIFNγ and their L-sel⁻ phenotype provide three independent lines ofevidence that a potentially pathogenic (Ando, et al. Cell Immunol.,124:132-143, 1989) Th1 type T-cell response is spontaneously primed toGAD in vivo early in NOD development.

EXAMPLE 8 CHARACTERIZATION OF GAD SPECIFIC T-CELL DETERMINANTRECOGNITION

The fine specificity of the anti-GAD T-cell response was mapped using aset of 38 peptides (numbered successively from the N-terminus) that were20-23 amino acids (aa) long and span the entire GAD₆₅ (Bu, et al., Proc.Natl. Acad. Sci., 89:2115-2119, 1992) sequence with 5aa overlaps (FIG.10).

Spleen cells were tested from 4 (FIG. 10a), 5 (FIG. 10b) and 7 (FIG.10c) week old NOD mice for proliferative responses (as described inExample 4) to the GAD peptides. Peptides were present in cultures at 7μM and the label was added during the last 16 hours of a 5-day culture.The peptides were synthesized using standard Fmoc chemistry and purifiedby reverse phase HPLC (Advanced Chemtech). The sequence of stimulatorypeptides are shown below in Table 9.

                                      TABLE 9                                     __________________________________________________________________________    Peptide Number                                                                         GAD Region                                                                           GAD Region    Amino Acid Sequence                             __________________________________________________________________________    6         78-97 KPCSCSKVDVNYAFLHATDL                                                                        SEQ ID NO:06                                    17       247-266                                                                              NMYAMMIARFKMFPEVKEKG                                                                        SEQ ID NO:07                                    23       335-356                                                                              TAGTTVYGAFDPLLAVADICKK                                                                      SEQ ID NO:08                                    32       479-498                                                                              EYLYNIIKNREGYEMVFDGK                                                                        SEQ ID NO:09                                    34       509-528                                                                              IPPSLRTLEDNEERMSRLSK                                                                        SEQ ID NO:10                                    35       524-543                                                                              SRLSKVAPVIKARMMEYGTT                                                                        SEQ ID NO:11                                    36       539-558                                                                              EYGTTMVSYQPLGDKVNFFR                                                                        SEQ ID NO:12                                    38       566-585                                                                              ATHQDIDFLIEEIERLGQDL                                                                        SEQ ID NO:13                                    __________________________________________________________________________

Murine and human GAD₆₅ are 95% identical at the amino acid level(555/585) and are 98% conserved, with most of the differences localizednear their N-termini. The underlined amino acid in the stimulatorypeptide sequences above are conservatively substituted in murine GAD₆₅.In separate experiments, the murine form of key peptides (#17 and #34)were tested and produced similar results.

As shown in FIG. 10, peptides that triggered stimulation indices >3 areindicated as black bars. These peptides did not induce proliferation inT-cells from NOD mice <3 or >16 weeks in age, or from control(BALB/c×NOD)F1 mice (data not shown). The data are represented as themean SI±standard error calculated from 3-6 individual mice tested twicein each age group. Characteristic results for peptide inducedblastogenesis in individual mice are shown in Table 6. The firstdetectable response, at 4 weeks of age, was confined to thecarboxy-terminal region of GAD, and involved two adjacent peptides (aa509-528 and 524-543, peptides #34 and #35, respectively, FIG. 10a). At 5weeks of age, responses to an additional determinant (aa 247-266,peptide #17, which contains a region of sequence similarity withCoxsackievirus (Kaufman, et al., J. Clin. Invest., 89:283-292, 1992)(FIG. 10b) were regularly recorded. During the next two weeks, responsesto peptide #17 (aa247-266) increased and T-cell autoimmunity spread totwo additional peptides at the carboxy terminus (aa 479-498 and 539-558;peptides #32 and #36 respectively, FIG. 10c). Subsequently, reactivityto the GAD peptides declined (data not shown), paralleling the loss ofresponse to the whole protein (FIG. 8). It is unclear why the initialT-cell response to βCAs fades in NOD mice. Possible explanationsinclude: a) immune regulatory mechanisms; b) exhaustion of the responsedue to the continuous stimulation by the endogenous antigen; and c)induction of anergy in specific T-cells owing to their recognition ofthe autoantigen on "non professional" antigen presenting cells such asthe β cells themselves (Markmann, et al., Nature, 336:476-479, 1988).

The gradual diversification of the primed autoreactive T-cell repertoirethat was observed in this naturally occurring autoimmune diseaseparallels the shifts in T-cell recognition recently observed inexperimentally induced autoimmunity to the CNS where autoreactivityspreads both intra- and intermolecularly among CNS proteins (Lehmannn,et al., Nature, 358:155-157, 1992; Perry, et al., J. Neuroimmunol.,33:7-15, 1991; Watanabe, et al., Nature, 305:150-153,1983; Liebert, etal., J. Neuroimmunol., 17:103-118, 1988). Apparently, lymphokinesecretion by the first wave of autoantigen specific T-cells in thetarget organ results in up-regulation of antigen presentation andcreates a microenvironment that favors priming of additionalautoreactive T-cells (Lehmann, et al., Immunol. Today, 14:203-208, 1993;Sarvetnick, et al., Nature, 346:844-847, 1990; Heath, et al., Nature,359:547-549, 1992). Since hsp reactive CD4+ T-cells are capable ofinducing IDDM (Elias, et al., Proc. Natl. Acad. Sci., 87:1576-1580,1990;Elias, et al., Proc. Natl. Acad. Sci., 88:3088-3091, 1991), theirrecruitment into the activated T-cell pool, along with T-cells reactiveto other βCAs, probably reflects an amplificatory cascade thateventually leads to β cell destruction.

In summary, the data above establish GAD as a critical target antigen inthe pathogenesis of IDDM in NOD mice. The results show that T-cellresponses to βCAs diversify both intramolecularly and intermolecularlyas the disease progresses, consistent with a dynamic autoimmunerepertoire (Lehmann, et al., Immunol. Today, 14:203-208, 1993). However,interference with the early autoreactive T-cell population can preventthe recruitment of additional autoantigens into the primed repertoirethereby halting a cascade of autoimmune responses that eventually leadsto β cell destruction. As a similar autoimmune progression is alsolikely to occur during the development of human IDDM (Palmer, J. P.,Predicting IDDM, Diabetes Reviews, 1:14-115, 1993; Atkinson, et al.,Lancet, 339:458459, 1992), these findings suggest that peptide-basedimmunotherapeutic agents would be useful in predicting and amelioratinghuman IDDM.

EXAMPLE 9 AUTOANTIBODY REACTIVITY WITH GAD FRAGMENTS

This example describes a study which examined the variability inrecognition of epitopes in human GAD₆₅ polypeptides by IDDMautoantibodies in sera of human patients.

Portions of human GAD₆₅ cDNA were amplified by the polymerase chainreaction (PCR; Saiki, et al., Science, 239:487, 1988) to produce DNAsegments encoding three polypeptide segments: amino acid residues 1-224(segment A); 224-398 (segment B); and 398-585 (segment C). Eachconstruct also contained a T₇ promoter, a consensus sequence for theinitiation of translation and an initiating methionine codon (Korak, M.,J. Cell Biol., 108:229, 1989). Each PCR product was then trascribed invitro with T₇ RNA polymerase and translated in vitro in a rabbitreticulocyte cell-free system in the presence of ³⁵ S-methionine, usingconditions recommended by the supplier (Amersham Corp., ArlingtonHeights, Ill.). Each test serum (30 μl) was incubated with the resulting³⁵ S labeled-polypeptides. The bound peptides were isolated with PAS andanalyzed by SDS-PAGE in 12% polyaacrylamide and autoradiography.

                  TABLE 10                                                        ______________________________________                                        IDDM PATIENT SERA REACTIVITY WITH GAD SEGMENTS                                           SEGMENT                                                            PATIENT      A            B     C                                             ______________________________________                                        Control (N = 7)                                                                            -            -     -                                             052          -            +     +                                             723          -            -     -                                             705          -            +     +                                             UC2          -            +     +                                             N. L.        -            -     -                                             L. I.        -            -     -                                             T. L.        -            -     -                                             P. T.        -            +     -                                             J. D.        -            -     -                                             B. Y.        -            +     +                                             M. C.        -            -     -                                             R. S.        -            -     -                                             K. O.        -            -     -                                             T. B.        -            -     -                                             S. M.        -            -     -                                             A. W.        -            +     -                                             J. B.        -            +     +                                             J. A.        -            -     -                                             P. C.        -            +     +                                             L. R.        -            -     -                                             J. M.        -            +     -                                             G. A.        -            -     -                                             ______________________________________                                    

As shown in Table 10, none of the specimens had detectable levels ofantibodies to the amino terminal third (segment A) of GAD whereas 9patients (41%) had antibodies reactive with the middle third (segment B)and 6 patients (27%) had antibodies to the carboxyl-terminal third(segment C) of GAD.

EXAMPLE 10 PREDICTION OF INCIPIENT IDDM BY GAD EPITOPE RECOGNITIONPATTERN

The increasing likelihood of an IDDM interventive therapy and the(recently acknowledged) benefits of managed glucose homeostasis inpreventing IDDM associated complications makes the early detection of βcell autoimmunity before clinical IDDM onset and in NIDDM patients (10%of whom eventually convert to IDDM) a crucial goal. Autoantibodies toGAD may provide the earliest and most reliable marker of impending IDDMamong the molecularly defined IDDM associated autoantigens. To determinewhether GAD peptides will bind to IDDM associated autoantibodies thefollowing study was conducted.

A set of peptides (20-23 amino acids in length, with 5 aa overlaps) thatspan the human GAD65 molecule were synthesized to determine whether serafrom most individuals at risk, pre-IDDM and with IDDM (in contrast tohealthy controls) do in fact produce antibodies that differentiallyrecognize GAD65 linear epitopes distributed throughout the molecule.

Patient sera and most control sera were those used in a previous study(Kaufman, et al., J. Clin. Investigation, supra) All samples were codedand tested in a blind manner. Peptides were synthesized using anautomatic instrument (Applied Biosystems, Foster City, Calif.) andstandard conditions. Peptides were dissolved in 60 mM sodium bicarbonatebuffer (pH 9.6) at 20 ug/mi and 100 ul of each was added to duplicatedwells of a 96 well Nunc-Immuno Plate. Peptides were allowed to bind at4° C. overnight. The plates were then washed three times with PBS+0.1%Tween 20 (wash buffer), after which the plates were pre-absorbed with 3%BSA in sodium bicarbonate buffer for 0.5 hours at 37° C., or at roomtemperature overnight. The plates were then washed 5 times with theabove wash buffer. 100 ul of serum at a 1/300 dilution in PBS+0.1% Tween20 and 1% BSA was added to each well and antibodies were allowed to bindfor 1 hour at 37° C. The plates were washed 5 times with wash buffer.100 ul of a 1/600 dilution of HRP-goat anti-human IgG (BRL,Gaithersberg, Md.) was added to each well and allowed to bind for 1 hourat 37° C. The plates were then washed 7 times and 100 ul of substratebuffer was added to each well for 30 minutes at room temperature. Thecolor development was measured at 410 nm using an ELISA plate reader(ICN, Biomedicals, Costa Mesa, Calif.). Positive sera were defined as:OD₄₁₀ of the sample/negative control ≦3.0.

The data shown in Table 11 establish that a number of GAD peptides wererecognized by patients previously shown to be 64 K positive, but not bycontrol sera. Each patient showed a different pattern of GAD epitoperecognition. Peptides 20, 21 and 25, were each recognized by 6/8patients, and none of the controls-with the exception of peptide 25which was recognized by 1 out of 13 controls. Based on immunoreactivityto 2 of these peptides (#20 and 21) 7/8 (88%) of the patients (and noneof the controls) could be identified as possessing GAD autoantibodies.Peptides 3, 6, 22, 25 and 37 were each recognized by only 25-37% of thepatients (and none of the control sera), but taken together, 75% of thepatients recognized at least one of these. Peptides 5, 9 and 24 wereoften positive for immunoreactivity by both control and patient sera.

This level of sensitivity is comparable to the best currently availableassays using whole GAD65 purified from brain or recombinant organisms.Besides avoiding laborious antigen purification, peptide basedautoantibody screening, together with PCR based HLA typing, may revealepitope recognition patterns associated with progression or lack ofprogression to IDDM and its associated complications. Individualsdetermined to be at high risk could then consider therapeuticintervention.

It should also be noted that the GAD peptides recognized byautoantibodies were different from those recognized by NOD GAD reactiveT cells in Example 6.

                                      TABLE 11                                    __________________________________________________________________________    EPITOPE RECOGNIATION OF HUMAN GAD65 PEPTIDES                                  __________________________________________________________________________    PEPTIDE                                                                            1 2 3 4 5 6 7 8 9 10                                                                              11                                                                              12                                                                              13                                                                              14                                                                              15                                                                              16                                                                              17                                                                              18                                                                              19                                   __________________________________________________________________________    Controls                                                                      A.P.         x       x                                                        P.T.         x                                                                5748         x       b                                                        5620         x       b                                                        4380         x       b                                                        S.M.         x       x                                                        FA8                                                                           FA12                                                                          1688         b                                                                FA9          x                                                                H.B          b                                                                FA2          x                                                                FA11         x                                                                                     2                                                        IDDM                                                                          At Risk                                                                       052    b     x x     b         b b b      x                                   825          x           x                                                    356          x       x                                                        L.I.     b   x     x     b                                                    Pre-IDDM                                                                      J.A.         x                                                                723      x   x       x                                                        P.T.                                                                          (1)      x   x       b                                                        (2)      b   x   b b b                                                        J.B.                                                                          (1)          b   b       b                                                    (2)      b   x   b b     b                                                    R.S.                                                                          (1)      x   x       b   b                                                    (2)      x   x b   b b   b                                                    B.Y.                                                                          (1)          x           x                                                    (2)          x                                                                J.B.                                                                          (1)          b   b       b                                                    (2)      b   x   b b     b                                                    At Onset                                                                      705          x     x                                                          S.H.   b x   x                                                                291      b x x   b                                                            048      x   x x     x                                                                 5 1   2   2 3   2                1                                   __________________________________________________________________________         20                                                                              21                                                                              22                                                                              23                                                                              24                                                                              25                                                                              26                                                                              27                                                                              28                                                                              29                                                                              30                                                                              31                                                                              32                                                                              33                                                                              34                                                                              35                                                                              36                                                                              37 38                                  __________________________________________________________________________    Controls                                                                      A.P.         x                                                                P.T.         x                                                                5748         x                                                                5620         x   b       b                                                    4380         x               b                                                S.M.   b     x x   b                                                          FA8          x                                                                FA12         x b                                                              1688         x                                                                FA9          x                                                                H.B          x                                                                FA2          x                                                                FA11         x                                                                               1                                                              IDDM                                                                          At Risk                                                                       052  x       x       b x   b              b                                   825          x b                                                              356    x     x x                                                              L.I. b       x               b                                                Pre-IDDM                                                                      J.A.   x     x                                                                723  x x     x x                   x   x                                      P.T.                                                                          (1)  x b     x b       x     b                                                (2)  x       x x       x     b   b                                            J.B.                                                                          (1)          x                                                                (2)  b b     x b       b                                                      R.S.                                                                          (1)          x x                                                              (2)  b       x x                                                              B.Y.                                                                          (1)          x                                                                (2)          x                                                                J.B.                                                                          (1)          x                                                                (2)  b b     x b       b                                                      At Onset                                                                      705  x x x   x x       x             x x                                      S.H. x x     x x                          b                                   291  x x x   x x   x         b   b                                            048  x x     x x       x     x   x        x                                        7 7 2     8   1   4     1   1 1 1 2  1                                   __________________________________________________________________________     x = positive for immunoreactivity as defined by OD/background 3.0             b = borderline responsive (OD/background ?2.5-2.9).                      

EXAMPLE 11 GAD IMMUNIZATION PROTECTS NOD MICE FROM IDDM

The availability of cDNAs encoding GAD65 allows the testing of thismolecule in new interventive therapies designed to interfere withGAD-specific T cells. Tests were conducted to examine the ability ofGAD65 immunization to protect NOD mice at 8 weeks of age, a time atwhich T cell responses to a number of b cell antigens and insulitis isalready well established. If GAD immunotherapy was effective at thisstage, it would hold promise for treatment in humans in which theautoimmune process has already been established.

METHODS

Antigens

An IPTG inducible T7 expression vector was used to express both humanGAD65 and E. coli b galactosidase (β-gal). In IPTG induced recombinantE. coli, GAD and β-gal constitute about 10-20% of the total bacterialprotein. However, almost all of the GAD was in inclusion bodies, whichcould be isolated and extensively washed to obtain material that isabout 80% GAD. We then did affinity purifications of GAD and β-gal onthe basis of a hexa-histidine "tag" which was attached to GAD during thesubcloning process. These extra histidine residues allow the rapidaffinity purification (Novagen) of GAD by metal affinity chromatography(Hochuli, et al., Bio Technology, 6:1321-1325, 1988). The inclusion bodymaterial is solubilized in 6M guanidine hydrochloride (GHCL), 10 mmβ-mercaptoethanol and 1% triton X-100. After binding to the column, thecolumn was extensively washed with GHCL and 8M urea in phosphatebuffers. Only the central peak GAD fraction was utilized for subsequentstudies. Human GAD65 shares 96% amino acid sequence identity with murineGAD65, with most of the amino acid differences being conservativesubstitutions.

The GAD preparation appeared to be free of immunologically detectablecontaminants. It also appeared to be free of bacterial contaminants onoverloaded silver stained gels. Analysis by a national referencelaboratory found <0.06 ng LPS/ug GAD. Human GAD65 did not induce T cellproliferation in <4 or >16 week old NOD or control BALB/c or(NOD/BALB/c) F1 spleen cells. The results using synthetic GAD peptides(FIG. 10) precisely parallel the data using whole recombinant GAD (FIG.8). Other antigens described herein elsewhere that are not involved inIDDM (such as the beta galactosidase) did not induce NOD T cellresponses. After immunizing mice with GAD, we were unable to detectcross reactive T cell responses in recall experiments with otherproteins that were purified from recombinant E. coli by the same metalaffinity chromatography procedure. Amino acid sequence analysis of GADand β-gal each gave a single expected amino acid N-terminal sequence. Ifthere had been appreciable endotoxins, heat shock proteins, or othercontaminants present in the GAD preparation, spleen, PBMC (Atkinson, etal.), and T cell proliferation responses that were not disease specificwould have been expected.

Breeder mice were purchased from Taconic Farms and housed under specificpathogen-free conditions. Only female NOD mice were used in this study.The average age of IDDM onset in unrelated females in the colony was 22weeks. Insulitis is generally observed beginning at 4 weeks of age. Tcell responses to GAD, HSP, CPH were found by 6 weeks in age. Theincidence of IDDM in female mice is 70-90% by one year of age.

Immunizations

At 8 weeks of age, 25 ug GAD or control β-gal. was injectedintraperitoneally (ip) in 100 ul of incomplete Freunds adjuvant (IFA).Because there may be a requirement for continual antigen presentation(Ramsdell, et al., Science, 257:1130-1133, 1992) mice were treated againevery 6 weeks. Urine glucose levels were monitored twice weekly. Afterobserving above normal glucose in urea, blood glucose levels weremonitored twice weekly. Two consecutive blood glucose level readings of300 mg/ml was considered as IDDM onset, after which the mice weresacrificed and spleen cells were tested as described above in Example 6for evidence of spleen cell proliferation.

Immunization of 8 week old NOD mice produced a clear delay in the onsetof IDDM compared to control β-gal immunized mice (FIG. 11). While two ofthe GAD immunized mice (open circles) developed IDDM at about the normalage of onset (20 weeks), the other 8 GAD immunized mice showed no signsof hyperglycemia until 36 weeks in age. Four of the GAD treated micedeveloped IDDM between 37 and 40 weeks in age. Four of the GAD treatedmice currently remain disease free (at 52 weeks of age). In contrast,the majority of β-gal injected mice (closed circles) had hyperglycemiaby 22 weeks of age and 6/10 developed IDDM by 27 weeks in age. At 52weeks of age, 2 of the β-gal treated mice remain disease free. Thisexperiment shows that GAD immunization significantly delayed (<0.02) orprevented diabetes of NOD mice in which β cell autoimmunity has alreadysignificantly progressed.

β cell autoimmunity is already well established at 8 weeks of age, andit is likely to also be in individuals determined to be at risk for IDDMon the basis of circulating autoantibodies. Although the mechanism ofthis protection is not clear, periodic injections of GAD have a profoundmoderating effect on the induction of disease.

                  TABLE 12                                                        ______________________________________                                        AMINO ACID SEQUENCES FOR GAD65                                                ______________________________________                                        1   SEQ ID NO:14                                                                              MASPGSGFWSFGSEDGSGDS                                          2   SEQ ID NO:15                                                                              GSGDSENPGTARAWCQVAQKFTG                                       3   SEQ ID NO:16                                                                              QKFTGGIGNKLCALLYGD                                            4   SEQ ID NO:17                                                                              LLYGDAEKPAESGGSQPPRA                                          5   SEQ ID NO:18                                                                              QPPRAAARKAACACDQKPCSC                                         6   SEQ ID NO:19                                                                              KPCSCSKVDVNYAFLHATDL                                          7   SEQ ID NO:20                                                                              HATDLLPACDGERPTLAFLQ                                          8   SEQ ID NO:21                                                                              LAFLQDVMNILLQYVVKSFDRS                                        9   SEQ ID NO:22                                                                              SFDRSTKVIDFHYPNELLQE                                          10  SEQ ID NO:23                                                                              ELLQEYNWELADQPQNLEEILM                                        11  SEQ ID NO:24                                                                              EEILMHCQTTLKYAIKTGHP                                          12  SEQ ID NO:25                                                                              KYGHPRYFNQLSTGLDMVGL                                          13  SEQ ID NO:26                                                                              DMVGLAADWLTSTANTNMFT                                          14  SEQ ID NO:27                                                                              TNMFTYEIAPVFVLLEYVTL                                          15  SEQ ID NO:28                                                                              EYVTLKKMREIIGWPGGSGD                                          16  SEQ ID NO:29                                                                              GGSGDGIFSPGGAISNMYAM                                          17  SEQ ID NO:30                                                                              NMYAMMIARFKMFPEVKEKG                                          18  SEQ ID NO:31                                                                              PEVKEKGMAALPRLIAFTSE                                          19  SEQ ID NO:32                                                                              AFTSEHSHFSLKKGAAALGI                                          20  SEQ ID NO:33                                                                              AALGIGTDSVILIKCDERGK                                          21  SEQ ID NO:34                                                                              DERGKMIPSDLERRILEAKQ                                          22  SEQ ID NO:35                                                                              LEAKQKGFVPFLVSATAGTT                                          23  SEQ ID NO:36                                                                              TAGTTVYGAFDPLLAVADICKK                                        24  SEQ ID NO:37                                                                              DICKKYKIWMHVDAAQGGGLLMS                                       25  SEQ ID NO:38                                                                              GLLMSRKHKWKLSGVERANS                                          26  SEQ ID NO:39                                                                              ERANSVTWNPHKMMGVPLQC                                          27  SEQ ID NO:40                                                                              VPLQCSALLVREEGLMQNCNQ                                         28  SEQ ID NO:41                                                                              QNCNQMHASYLFQQDKHYDL                                          29  SEQ ID NO:42                                                                              KHYDLSYDTGDKALQCGRHV                                          30  SEQ ID NO:43                                                                              CGRHVDVFKLWLMWRAKGTTG                                         31  SEQ ID NO:44                                                                              KGTTGFEAHVDKCLELAEYLYN                                        32  SEQ ID NO:45                                                                              EYLYNIIKNREGYEMVFDGK                                          33  SEQ ID NO:46                                                                              VFDGKPQHTNVCFWYIPPSL                                          34  SEQ ID NO:47                                                                              IPPSLRTLEDNEERMSRLSK                                          35  SEQ ID NO:48                                                                              SRLSKVAPVIKARMMEYGTT                                          36  SEQ ID NO:49                                                                              EYGTTMVSYQPLGDKVNFFR                                          37  SEQ ID NO:50                                                                              VNFFRMVISNPAATHQDIDF                                          38  SEQ ID NO:51                                                                              ATHQDIDFLIEEIERLGQDL                                          ______________________________________                                    

The invention now being fully described, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade without departing from the scope of the invention.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 60                                            - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 23 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - Ser Ile Ala Ala Arg Tyr Lys Tyr Phe Pro Gl - #u Val Lys Thr Lys Gly         #                15                                                           - Met Ala Ala Val Pro Lys Leu                                                             20                                                                - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 24 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - Ala Met Met Ile Ala Arg Phe Lys Met Phe Pr - #o Glu Val Lys Glu Lys         #                15                                                           - Gly Met Ala Ala Leu Pro Arg Leu                                                         20                                                                - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 23 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 - Phe Ile Glu Trp Leu Lys Val Lys Ile Leu Pr - #o Glu Val Lys Glu Lys         #                15                                                           - His Glu Phe Leu Ser Arg Leu                                                             20                                                                - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 15 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 - Met Lys Ser Met Cys Pro Gln Ala Gln Leu Ly - #s Val Lys Tyr Leu             #                15                                                           - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 16 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 - Ala Arg Phe Lys Met Phe Pro Glu Val Lys Gl - #u Lys Gly Met Ala Ala         #                15                                                           - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 20 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 - Lys Pro Cys Ser Cys Ser Lys Val Asp Val As - #n Tyr Ala Phe Leu His         #                15                                                           - Ala Thr Asp Leu                                                                         20                                                                - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 20 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             #ID NO:7: (xi) SEQUENCE DESCRIPTION: SEQ                                      - Asn Met Tyr Ala Met Met Ile Ala Arg Phe Ly - #s Met Phe Pro Glu Val         #                15                                                           - Lys Glu Lys Gly                                                                         20                                                                - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 22 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 - Thr Ala Gly Thr Thr Val Tyr Gly Ala Phe As - #p Pro Leu Leu Ala Val         #                15                                                           - Ala Asp Ile Cys Lys Lys                                                                 20                                                                - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 20 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 - Glu Tyr Leu Tyr Asn Ile Ile Lys Asn Arg Gl - #u Gly Tyr Glu Met Val         #                15                                                           - Phe Asp Gly Lys                                                                         20                                                                - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 20 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                - Ile Pro Pro Ser Leu Arg Tyr Leu Glu Asp As - #n Glu Glu Arg Met Ser         #                15                                                           - Arg Leu Ser Lys                                                                         20                                                                - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 20 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                - Ser Arg Leu Ser Lys Val Ala Pro Val Ile Ly - #s Ala Arg Met Met Glu         #                15                                                           - Tyr Gly Thr Thr                                                                         20                                                                - (2) INFORMATION FOR SEQ ID NO:12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 20 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                - Glu Tyr Gly Thr Thr Met Val Ser Tyr Gln Pr - #o Leu Gly Asp Lys Val         #                15                                                           - Asn Phe Phe Arg                                                                         20                                                                - (2) INFORMATION FOR SEQ ID NO:13:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 20 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                - Ala Thr His Gln Asp Ile Asp Phe Leu Ile Gl - #u Glu Ile Glu Arg Leu         #                15                                                           - Gly Gln Asp Leu                                                                         20                                                                - (2) INFORMATION FOR SEQ ID NO:14:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 20 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                - Met Ala Ser Pro Gly Ser Gly Phe Trp Ser Ph - #e Gly Ser Glu Asp Gly         #                15                                                           - Ser Gly Asp Ser                                                                         20                                                                - (2) INFORMATION FOR SEQ ID NO:15:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 23 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                - Gly Ser Gly Asp Ser Glu Asn Pro Gly Thr Al - #a Arg Ala Trp Cys Gln         #                15                                                           - Val Ala Gln Lys Phe Thr Gly                                                             20                                                                - (2) INFORMATION FOR SEQ ID NO:16:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 20 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                - Gln Lys Phe Thr Gly Gly Ile Gly Ile Gly As - #n Lys Leu Cys Ala Leu         #                15                                                           - Leu Tyr Gly Asp                                                                         20                                                                - (2) INFORMATION FOR SEQ ID NO:17:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 20 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                - Leu Leu Tyr Gly Asp Ala Glu Lys Pro Ala Gl - #u Ser Gly Gly Ser Gln         #                15                                                           - Pro Pro Arg Ala                                                                         20                                                                - (2) INFORMATION FOR SEQ ID NO:18:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 21 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                - Gln Pro Pro Arg Ala Ala Ala Arg Lys Ala Al - #a Cys Ala Cys Asp Gln         #                15                                                           - Lys Pro Cys Ser Cys                                                                     20                                                                - (2) INFORMATION FOR SEQ ID NO:19:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 20 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                - Lys Pro Cys Ser Cys Ser Lys Val Asp Val As - #n Tyr Ala Phe Leu His         #                15                                                           - Ala Thr Asp Leu                                                                         20                                                                - (2) INFORMATION FOR SEQ ID NO:20:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 20 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                - His Ala Thr Asp Leu Leu Pro Ala Cys Asp Gl - #y Glu Arg Pro Thr Leu         #                15                                                           - Ala Phe Leu Gln                                                                         20                                                                - (2) INFORMATION FOR SEQ ID NO:21:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 22 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                - Leu Ala Phe Leu Gln Asp Val Met Asn Ile Le - #u Leu Gln Tyr Val Val         #                15                                                           - Lys Ser Phe Asp Arg Ser                                                                 20                                                                - (2) INFORMATION FOR SEQ ID NO:22:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 20 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             #ID NO:22:(xi) SEQUENCE DESCRIPTION: SEQ                                      -      Ser Phe Asp Arg Ser Thr Lys Val - # Ile Asp Phe His Tyr Pro Asn        Glu                                                                           #   15                                                                        -      Leu Leu Gln Glu                                                                         20                                                           - (2) INFORMATION FOR SEQ ID NO:23:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 22 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             #ID NO:23:(xi) SEQUENCE DESCRIPTION: SEQ                                      -      Glu Leu Leu Gln Glu Tyr Asn Trp - # Glu Leu Ala Asp Gln Pro Gln        Asn                                                                           #   15                                                                        -      Leu Glu Glu Ile Leu Met                                                                 20                                                           - (2) INFORMATION FOR SEQ ID NO:24:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 20 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             #ID NO:24:(xi) SEQUENCE DESCRIPTION: SEQ                                      -      Glu Glu Ile Leu Met His Cys Gln - # Thr Thr Leu Lys Tyr Ala Ile        Lys                                                                           #   15                                                                        -      Thr Gly His Pro                                                                         20                                                           - (2) INFORMATION FOR SEQ ID NO:25:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 20 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             #ID NO:25:(xi) SEQUENCE DESCRIPTION: SEQ                                      -      Lys Tyr Gly His Pro Arg Tyr Phe - # Asn Gln Leu Ser Thr Gly Leu        Asp                                                                           #   15                                                                        -      Met Val Gly Leu                                                                         20                                                           - (2) INFORMATION FOR SEQ ID NO:26:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 20 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             #ID NO:26:(xi) SEQUENCE DESCRIPTION: SEQ                                      -      Asp Met Val Gly Leu Ala Ala Asp - # Trp Leu Thr Ser Thr Ala Asn        Thr                                                                           #   15                                                                        -      Asn Met Phe Thr                                                                         20                                                           - (2) INFORMATION FOR SEQ ID NO:27:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 20 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             #ID NO:27:(xi) SEQUENCE DESCRIPTION: SEQ                                      -      Thr Asn Met Phe Thr Tyr Glu Ile - # Ala Pro Val Phe Val Leu Leu        Glu                                                                           #   15                                                                        -      Tyr Val Thr Leu                                                                         20                                                           - (2) INFORMATION FOR SEQ ID NO:28:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 20 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             #ID NO:28:(xi) SEQUENCE DESCRIPTION: SEQ                                      -      Glu Tyr Val Thr Leu Lys Lys Met - # Arg Glu Ile Ile Gly Trp Pro        Gly                                                                           #   15                                                                        -      Gly Ser Gly Asp                                                                         20                                                           - (2) INFORMATION FOR SEQ ID NO:29:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 20 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             #ID NO:29:(xi) SEQUENCE DESCRIPTION: SEQ                                      -      Gly Gly Ser Gly Asp Gly Ile Phe - # Ser Pro Gly Gly Ala Ile Ser        Asn                                                                           #   15                                                                        -      Met Tyr Ala Met                                                                         20                                                           - (2) INFORMATION FOR SEQ ID NO:30:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 20 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             #ID NO:30:(xi) SEQUENCE DESCRIPTION: SEQ                                      -      Asn Met Tyr Ala Met Met Ile Ala - # Arg Phe Lys Met Phe Pro Glu        Val                                                                           #   15                                                                        -      Lys Glu Lys Gly                                                                         20                                                           - (2) INFORMATION FOR SEQ ID NO:31:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 20 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             #ID NO:31:(xi) SEQUENCE DESCRIPTION: SEQ                                      -      Pro Glu Val Lys Glu Lys Gly Met - # Ala Ala Leu Pro Arg Leu Ile        Ala                                                                           #   15                                                                        -      Phe Thr Ser Glu                                                                         20                                                           - (2) INFORMATION FOR SEQ ID NO:32:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 20 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             #ID NO:32:(xi) SEQUENCE DESCRIPTION: SEQ                                      -      Ala Phe Thr Ser Glu His Ser His - # Phe Ser Leu Lys Lys Gly Ala        Ala                                                                           #   15                                                                        -      Ala Leu Gly Ile                                                                         20                                                           - (2) INFORMATION FOR SEQ ID NO:33:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 20 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             #ID NO:33:(xi) SEQUENCE DESCRIPTION: SEQ                                      -      Ala Ala Leu Gly Ile Gly Thr Asp - # Ser Val Ile Leu Ile Lys Cys        Asp                                                                           #   15                                                                        -      Glu Arg Gly Lys                                                                         20                                                           - (2) INFORMATION FOR SEQ ID NO:34:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 20 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             #ID NO:34:(xi) SEQUENCE DESCRIPTION: SEQ                                      -      Asp Glu Arg Gly Lys Met Ile Pro - # Ser Asp Leu Glu Arg Arg Ile        Leu                                                                           #   15                                                                        -      Glu Ala Lys Gln                                                                         20                                                           - (2) INFORMATION FOR SEQ ID NO:35:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 20 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             #ID NO:35:(xi) SEQUENCE DESCRIPTION: SEQ                                      -      Leu Glu Ala Lys Gln Lys Gly Phe - # Val Pro Phe Leu Val Ser Ala        Thr                                                                           #   15                                                                        -      Ala Gly Thr Thr                                                                         20                                                           - (2) INFORMATION FOR SEQ ID NO:36:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 22 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             #ID NO:36:(xi) SEQUENCE DESCRIPTION: SEQ                                      -      Thr Ala Phe Thr Thr Val Tyr Gly - # Ala Phe Asp Pro Leu Leu Ala        Val                                                                           #   15                                                                        -      Ala Asp Ile Cys Lys Lys                                                                 20                                                           - (2) INFORMATION FOR SEQ ID NO:37:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 23 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             #ID NO:37:(xi) SEQUENCE DESCRIPTION: SEQ                                      -      Asp Ile Cys Lys Lys Tyr Lys Ile - # Trp Met His Val Asp Ala Ala        Trp                                                                           #   15                                                                        -      Gly Gly Gly Leu Leu Met Ser                                                             20                                                           - (2) INFORMATION FOR SEQ ID NO:38:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 20 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             #ID NO:38:(xi) SEQUENCE DESCRIPTION: SEQ                                      -      Gly Leu Leu Met Ser Arg Lys His - # Lys Trp Lys Leu Ser Gly Val        Glu                                                                           #   15                                                                        -      Arg Ala Asn Ser                                                                         20                                                           - (2) INFORMATION FOR SEQ ID NO:39:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 20 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             #ID NO:39:(xi) SEQUENCE DESCRIPTION: SEQ                                      -      Glu Arg Ala Asn Ser Val Thr Trp - # Asn Pro His Lys Met Met Gly        Val                                                                           #   15                                                                        -      Pro Leu Gln Cys                                                                         20                                                           - (2) INFORMATION FOR SEQ ID NO:40:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 21 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             #ID NO:40:(xi) SEQUENCE DESCRIPTION: SEQ                                      -      Val Pro Leu Gln Cys Ser Ala Leu - # Leu Val Arg Glu Glu Gly Leu        Met                                                                           #   15                                                                        -      Gln Asn Cys Asn Gln                                                                     20                                                           - (2) INFORMATION FOR SEQ ID NO:41:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 20 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             #ID NO:41:(xi) SEQUENCE DESCRIPTION: SEQ                                      -      Gln Asn Cys Asn Gln Met His Ala - # Ser Tyr Leu Phe Gln Gln Asp        Lys                                                                           #   15                                                                        -      His Tyr Asp Leu                                                                         20                                                           - (2) INFORMATION FOR SEQ ID NO:42:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 20 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             #ID NO:42:(xi) SEQUENCE DESCRIPTION: SEQ                                      -      Lys His Tyr Asp Leu Ser Tyr Asp - # Thr Gly Asp Lys Ala Leu Gln        Cys                                                                           #   15                                                                        -      Gly Arg His Val                                                                         20                                                           - (2) INFORMATION FOR SEQ ID NO:43:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 21 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             #ID NO:43:(xi) SEQUENCE DESCRIPTION: SEQ                                      -      Cys Gly Arg His Val Asp Val Phe - # Lys Leu Trp Leu Met Trp Arg        Ala                                                                           #   15                                                                        -      Lys Gly Thr Thr Gly                                                                     20                                                           - (2) INFORMATION FOR SEQ ID NO:44:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 22 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             #ID NO:44:(xi) SEQUENCE DESCRIPTION: SEQ                                      -      Lys Gly Thr Thr Gly Phe Glu Ala - # His Val Asp Lys Cys Leu Glu        Leu                                                                           #   15                                                                        -      Ala Glu Tyr Leu Tyr Asn                                                                 20                                                           - (2) INFORMATION FOR SEQ ID NO:45:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 20 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             #ID NO:45:(xi) SEQUENCE DESCRIPTION: SEQ                                      -      Glu Tyr Leu Tyr Asn Ile Ile Lys - # Asn Arg Glu Gly Tyr Glu Met        Val                                                                           #   15                                                                        -      Phe Asp Gly Lys                                                                         20                                                           - (2) INFORMATION FOR SEQ ID NO:46:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 20 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             #ID NO:46:(xi) SEQUENCE DESCRIPTION: SEQ                                      -      Val Phe Asp Gly Lys Pro Gln His - # Thr Asn Val Cys Phe Trp Tyr        Ile                                                                           #   15                                                                        -      Pro Pro Ser Leu                                                                         20                                                           - (2) INFORMATION FOR SEQ ID NO:47:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 20 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             #ID NO:47:(xi) SEQUENCE DESCRIPTION: SEQ                                      -      Ile Pro Pro Ser Leu Arg Thr Leu - # Glu Asp Asn Glu Glu Arg Met        Ser                                                                           #   15                                                                        -      Arg Leu Ser Lys                                                                         20                                                           - (2) INFORMATION FOR SEQ ID NO:48:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 20 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             #ID NO:48:(xi) SEQUENCE DESCRIPTION: SEQ                                      -      Ser Arg Leu Ser Lys Val Ala Pro - # Val Ile Lys Ala Arg Met Met        Glu                                                                           #   15                                                                        -      Tyr Gly Thr Thr                                                                         20                                                           - (2) INFORMATION FOR SEQ ID NO:49:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 21 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             #ID NO:49:(xi) SEQUENCE DESCRIPTION: SEQ                                      -      Glu Tyr Gly Thr Thr Thr Met Val - # Ser Tyr Gln Pro Leu Gly Asp        Lys                                                                           #   15                                                                        -      Val Asn Phe Phe Arg                                                                     20                                                           - (2) INFORMATION FOR SEQ ID NO:50:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 20 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             #ID NO:50:(xi) SEQUENCE DESCRIPTION: SEQ                                      -      Val Asn Phe Phe Arg Met Val Ile - # Ser Asn Pro Ala Ala Thr His        Gln                                                                           #   15                                                                        -      Asp Ile Asp Phe                                                                         20                                                           - (2) INFORMATION FOR SEQ ID NO:51:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 20 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             #ID NO:51:(xi) SEQUENCE DESCRIPTION: SEQ                                      -      Ala Thr His Gln Asp Ile Asp Phe - # Leu Ile Glu Glu Ile Glu Arg        Leu                                                                           #   15                                                                        -      Gly Gln Asp Leu                                                                         20                                                           - (2) INFORMATION FOR SEQ ID NO:52:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 60 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             #ID NO:52:(xi) SEQUENCE DESCRIPTION: SEQ                                      -      Tyr Glu Ile Ala Pro Val Phe Val - # Leu Met Glu Gln Ile Thr Leu        Lys                                                                           #   15                                                                        -      Lys Met Arg Glu Ile Val Gly Trp - # Ser Ser Lys Asp Gly Asp Gly        Ile                                                                           #                 30                                                          -      Phe Ser Pro Gly Gly Ala Ile Ser - # Asn Met Tyr Ser Ile Met Ala        Ala                                                                           #             45                                                              -      Arg Tyr Lys Phe Phe Pro Glu Val - # Lys Thr Lys Gly                    #         60                                                                  - (2) INFORMATION FOR SEQ ID NO:53:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 53 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             #ID NO:53:(xi) SEQUENCE DESCRIPTION: SEQ                                      -      Tyr Glu Ile Ala Pro Val Phe Val - # Leu Leu Glu Tyr Val Arg Glu        Ile                                                                           #   15                                                                        -      Ile Gly Trp Pro Gly Gly Ser Asp - # Gly Ile Phe Ser Pro Gly Gly        Ala                                                                           #                 30                                                          -      Ile Ser Asn Tyr Ala Met Leu Ile - # Ala Arg Tyr Lys Met Phe Pro        Glu                                                                           #             45                                                              -      Val Lys Glu Lys Gly                                                             50                                                                   - (2) INFORMATION FOR SEQ ID NO:54:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 2026 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                #ID NO:54:(xi) SEQUENCE DESCRIPTION: SEQ                                      - GGGCGTGCGG GGTCGAGCCG AAGCAGCTTG CCCGCAGCCA CTCGGAGGCG AC - #CAGCGCCA       60                                                                            - GACTAGCAGA ACCCATGGCA TCTCCGGGCT CTGGCTTTTG GTCCTTCGGA TC - #TGAAGATG       120                                                                           - GCTCTGGGGA TCCTGAGAAC CCGGGAACAG CGAGAGCCTG GTGCCAGGTG GC - #CCAAAAGT       180                                                                           - TCACGGGCGG CATCGGAAAC AAGCTATGCG CTCTGCTCTA CGGAGACTCT GA - #GAAGCCAG       240                                                                           - CAGAGAGCGG CGGGAGCGTG ACCTCGCGGG CCGCCACTCG GAAGGTCGCC TG - #CACCTGTG       300                                                                           - ACCAAAAACC CTGCAGCTGC CCCAAAGGAG ATGTCAATTA TGCACTTCTC CA - #CGCAACAG       360                                                                           - ACCTGCTGCC AGCCTGTGAA GGAGAAAGGC CCACTCTCGC ATTTCTGCAA GA - #TGTAATGA       420                                                                           - ACATTTTGCT TCAGTACGTG GTGAAAAGTT TTGATAGATC AACTAAAGTG AT - #TGATTTCC       480                                                                           - ATTACCCCAA TGAGCTTCTT CAAGAGTATA ATTGGGAATT GGCAGACCAA CC - #GCAAAATC       540                                                                           - TGGAGGAAAT TTTGACGCAC TGCCAAACAA CTCTAAAATA TGCGATTAAA AC - #AGGGCATC       600                                                                           - CCCGATATTT TAATCAGCTG TCTACCGGAT TGGATATGGT TGGATTAGCA GC - #AGATTGGT       660                                                                           - TGACATCAAC AGCAAACACG AACATGTTTA CCTATGAGAT CGCCCCTGTA TT - #TGTACTAC       720                                                                           - TGGAATATGT GACACTAAAG AAAATGAGGG AAATCATTGG CTGGCCAGGA GG - #CTCTGGCG       780                                                                           - ATGGAATCTT TTCTCCTGGT GGTGCCATCT CCAACATGTA CGCCATGCTC AT - #TGCCCGCT       840                                                                           - ATAAGATGTT TCCAGAAGTC AAGGAAAAGG GGATGGCGGC GGTGCCCAGG CT - #CATCGCAT       900                                                                           - TCACGTCAGA GCATAGTCAC TTTTCTCTCA AGAAGGGAGC TGCAGCCTTG GG - #GATCGGAA       960                                                                           - CAGACAGCGT GATTCTGATT AAATGTGATG AGAGAGGGAA AATGATCCCA TC - #TGACCTTG       1020                                                                          - AAAGAAGAAT CCTTGAAGTC AAACAGAAAG GATTTGTTCC TTTCCTGGTG AG - #TGCCACAG       1080                                                                          - AAAGAAGAAT CCTTGAAGTC AAACAGAAAG GATTTGTTCC TTTCCTGGTG AG - #TGCCACAG       1140                                                                          - CTGGAACCAC TGTGTACGGG GCTTTTGATC CTCTCTTGGC TGTAGCTGAC AT - #CTGCAAAA       1200                                                                          - AATATAAGAT CTGGATGCAT GTGGATGCTG CTTGGGGTGG AGGGTTACTG AT - #GTCTCGGA       1260                                                                          - AACACAAGTG GAAGCTGAAC GGTGTGGAGA GGGCCAACTC TGTGACATGG AA - #TCCCCACA       1320                                                                          - AGATGATGGG TGTCCCCTTG CAATGTTCGG CTCTCCTGGT CAGAGAGGAG GG - #ACTGATGC       1380                                                                          - AGAGCTGCAA CCAGATGCAT GCTTCCTACC TCTTTCAGCA AGATAAGCAC TA - #TGACCTGT       1440                                                                          - CCTATGACAC GGGAGACAAG GCCTTGCAGT GTGGACGCCA CGTCGATGTC TT - #TAAATTAT       1500                                                                          - GGCTCATGTG GAGAGCAAAG GGGACTACTG GATTTGAAGC TCACATTGAT AA - #GTGTTTGG       1560                                                                          - AGCTGGCAGA GTATTTATAC AATATCATTA AAAACCGAGA AGGATATGAA AT - #GGTGTTCG       1620                                                                          - ATGGGAAGCC TCAGCACACA AATGTCTGCT TCTGGTTTGT ACCTCCTAGT TT - #GCGAGTTC       1680                                                                          - TGGAAGACAA TGAAGAGAGA ATGAGCCGCC TCTCAAAGGT GGCGCCAGTG AT - #TAAAGCCA       1740                                                                          - GAATGATGGA GTATGGGACC ACAATGGTCA GCTACCAACC CTTAGGAGAT AA - #GGTCAACT       1800                                                                          - TCTTCCGCAT GGTCATCTCA AACCCTGCAG CAACTCACCA AGACATTGAC TT - #CCTCATTG       1860                                                                          - AAGAAATCGA ACGCCTGGGA CAAGATTTGT AATCACTTTG CTCACCAAAC TT - #TCAGTTCT       1920                                                                          - CTAGGTAGAC AGCTAAGTTG TCACAAACTG TGTAAATGTA TTTGTAGTTT GT - #TCCAGAGT       1980                                                                          #             2026TCGTG GTGTCACAGT AGAGTCCAGT TTAAAA                          - (2) INFORMATION FOR SEQ ID NO:55:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 605 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             #ID NO:55:(xi) SEQUENCE DESCRIPTION: SEQ                                      -      Met Ala Ser Pro Gly Ser Gly Phe - # Trp Ser Phe Gly Ser Glu Asp        Gly                                                                           #   15                                                                        -      Ser Gly Asp Pro Glu Asn Pro Gly - # Thr Ala Arg Ala Trp Cys Gln        Val                                                                           #                 30                                                          -      Ala Gln Lys Phe Thr Gly Gly Ile - # Gly Asn Lys Leu Cys Ala Leu        Leu                                                                           #             45                                                              -      Tyr Gly Asp Ser Glu Lys Pro Ala - # Glu Ser Gly Gly Ser Val Thr        Ser                                                                           #         60                                                                  -      Arg Ala Ala Thr Arg Lys Val Ala - # Cys Thr Cys Asp Gln Lys Pro        Cys                                                                           #     80                                                                      -      Ser Cys Pro Lys Gly Asp Val Asn - # Tyr Ala Leu Leu His Ala Thr        Asp                                                                           #   95                                                                        -      Leu Leu Pro Ala Cys Glu Gly Glu - # Arg Pro Thr Leu Ala Phe Leu        Gln                                                                           #                110                                                          -      Asp Val Met Asn Ile Leu Leu Gln - # Tyr Val Val Lys Ser Phe Asp        Arg                                                                           #            125                                                              -      Ser Thr Lys Val Ile Asp Phe His - # Tyr Pro Asn Glu Leu Leu Gln        Glu                                                                           #        140                                                                  -      Tyr Asn Trp Glu Leu Ala Asp Gln - # Pro Gln Asn Leu Glu Glu Ile        Leu                                                                           #    160                                                                      -      Thr His Cys Gln Thr Thr Leu Lys - # Tyr Ala Ile Lys Thr Gly His        Pro                                                                           #   175                                                                       -      Arg Tyr Phe Asn Gln Leu Ser Thr - # Gly Leu Asp Met Val Gly Leu        Ala                                                                           #                190                                                          -      Ala Asp Trp Leu Thr Ser Thr Ala - # Asn Thr Asn Met Phe Thr Tyr        Glu                                                                           #            205                                                              -      Ile Ala Pro Val Phe Val Leu Leu - # Glu Tyr Val Thr Leu Lys Lys        Met                                                                           #        220                                                                  -      Arg Glu Ile Ile Gly Trp Pro Gly - # Gly Ser Gly Asp Gly Ile Phe        Ser                                                                           #    240                                                                      -      Pro Gly Gly Ala Ile Ser Asn Met - # Tyr Ala Met Leu Ile Ala Arg        Tyr                                                                           #   255                                                                       -      Lys Met Phe Pro Glu Val Lys Glu - # Lys Gly Met Ala Ala Val Pro        Arg                                                                           #                270                                                          -      Leu Ile Ala Phe Thr Ser Glu His - # Ser His Phe Ser Leu Lys Lys        Gly                                                                           #            285                                                              -      Ala Ala Ala Leu Gly Ile Gly Thr - # Asp Ser Val Ile Leu Ile Lys        Cys                                                                           #        300                                                                  -      Asp Glu Arg Gly Lys Met Ile Pro - # Ser Asp Leu Glu Arg Arg Ile        Leu                                                                           #    320                                                                      -      Glu Val Lys Gln Lys Gly Phe Val - # Pro Phe Leu Val Ser Ala Thr        Ala                                                                           #   335                                                                       -      Arg Arg Ile Leu Glu Val Lys Gln - # Lys Gly Phe Val Pro Phe Leu        Val                                                                           #                350                                                          -      Ser Ala Thr Ala Gly Thr Thr Val - # Tyr Gly Ala Phe Asp Pro Leu        Leu                                                                           #            365                                                              -      Ala Val Ala Asp Ile Cys Lys Lys - # Tyr Lys Ile Trp Met His Val        Asp                                                                           #        380                                                                  -      Ala Ala Trp Gly Gly Gly Leu Leu - # Met Ser Arg Lys His Lys Trp        Lys                                                                           #    400                                                                      -      Leu Asn Gly Val Glu Arg Ala Asn - # Ser Val Thr Trp Asn Pro His        Lys                                                                           #   415                                                                       -      Met Met Gly Val Pro Leu Gln Cys - # Ser Ala Leu Leu Val Arg Glu        Glu                                                                           #                430                                                          -      Gly Leu Met Gln Ser Cys Asn Gln - # Met His Ala Ser Tyr Leu Phe        Gln                                                                           #            445                                                              -      Gln Asp Lys His Tyr Asp Leu Ser - # Tyr Asp Thr Gly Asp Lys Ala        Leu                                                                           #        460                                                                  -      Gln Cys Gly Arg His Val Asp Val - # Phe Lys Leu Trp Leu Met Trp        Arg                                                                           #    480                                                                      -      Ala Lys Gly Thr Thr Gly Phe Glu - # Ala His Ile Asp Lys Cys Leu        Glu                                                                           #   495                                                                       -      Leu Ala Glu Tyr Leu Tyr Asn Ile - # Ile Lys Asn Arg Glu Gly Tyr        Glu                                                                           #                510                                                          -      Met Val Phe Asp Gly Lys Pro Gln - # His Thr Asn Val Cys Phe Trp        Phe                                                                           #            525                                                              -      Val Pro Pro Ser Leu Arg Val Leu - # Glu Asp Asn Glu Glu Arg Met        Ser                                                                           #        540                                                                  -      Arg Leu Ser Lys Val Ala Pro Val - # Ile Lys Ala Arg Met Met Glu        Tyr                                                                           #    560                                                                      -      Gly Thr Thr Met Val Ser Tyr Gln - # Pro Leu Gly Asp Lys Val Asn        Phe                                                                           #   575                                                                       -      Phe Arg Met Val Ile Ser Asn Pro - # Ala Ala Thr His Gln Asp Ile        Asp                                                                           #                590                                                          -      Phe Leu Ile Glu Glu Ile Glu Arg - # Leu Gly Gln Asp Leu                #            605                                                              - (2) INFORMATION FOR SEQ ID NO:56:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 2100 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                #ID NO:56:(xi) SEQUENCE DESCRIPTION: SEQ                                      - AGCTCGCCCG CAGCTCGCAC TCGCAGGCGA CCTGCTCCAG TCTCCAAAGC CG - #ATGGCATC       60                                                                            - TCCGGGCTCT GGCTTTTGGT CTTTCGGGTC GGAAGATGGC TCTGGGGATT CC - #GAGAATCC       120                                                                           - CGGCACAGCG CGAGCCTGGT GCCAAGTGGC TCAGAAGTTC ACGGGCGGCA TC - #GGAAACAA       180                                                                           - ACTGTGCGCC CTGCTCTACG GAGACGCCGA GAAGCCGGCG GAGAGCGGCG GG - #AGCCAACC       240                                                                           - CCCGCGGGCC GCCGCCCGGA AGGCCGCCTG CGCCTGCGAC CAGAAGCCCT GC - #AGCTGCTC       300                                                                           - CAAAGTGGAT GTCAACTACG CGTTTCTCCA TGCAACAGAC CTGCTGCCGG CG - #TGTGATGG       360                                                                           - AGAAAGGCCC ACTTTGGCGT TTCTGCAAGA TGTTATGAAC ATTTTACTTC AG - #TATGTGGT       420                                                                           - GAAAAGTTTC GATAGATCAA CCAAAGTGAT TGATTTCCAT TATCCTAATG AG - #CTTCTCCA       480                                                                           - AGAATATAAT TGGGAATTGG CAGACCAACC ACAAAATTTG GAGGAAATTT TG - #ATGCATTG       540                                                                           - CCAAACAACT CTAAAATATG CAATTAAAAC AGGGCATCCT AGATACTTCA AT - #CAACTTTC       600                                                                           - TACTGGTTTG GATATGGTTG GATTAGCAGC AGACTGGCTG ACATCAACAG CA - #AATACTAA       660                                                                           - CATGTTCACC TATGAAATTG CTCCAGTATT TGTGCTTTTG GAATATGTCA CA - #CTAAAGAA       720                                                                           - AATGAGAGAA ATCATTGGCT GGCCAGGGGG CTCTGGCGAT GGGATATTTT CT - #CCCGGTGG       780                                                                           - CGCCATATCT AACATGTATG CCATGATGAT CGCACGCTTT AAGATGTTCC CA - #GAAGTCAA       840                                                                           - GGAGAAAGGA ATGGCTGCTC TTCCCAGGCT CATTGCCTTC ACGTCTGAAC AT - #AGTCATTT       900                                                                           - TTCTCTCAAG AAGGGAGCTG CAGCCTTAGG GATTGGAACA GACAGCGTGA TT - #CTGATTAA       960                                                                           - ATGTGATGAG AGAGGGAAAA TGATTCCATC TGATCTTGAA AGAAGGATTC TT - #GAAGCCAA       1020                                                                          - ACAGAAAGGG TTTGTTCCTT TCCTCGTGAG TGCCACAGCT GGAACCACCG TG - #TACGGAGC       1080                                                                          - ATTTGACCCC CTCTTAGCTG TCGCTGACAT TTGCAAAAAG TATAAGATCT GG - #ATGCATGT       1140                                                                          - GGATGCAGCT TGGGGTGGGG GATTACTGAT GTCCCGAAAA CACAAGTGGA AA - #CTGAGTGG       1200                                                                          - CGTGGAGAGG GCCAACTCTG TGACGTGGAA TCCACACAAG ATGATGGGAG TC - #CCTTTGCA       1260                                                                          - GTGCTCTGCT CTCCTGGTTA GAGAAGAGGG ATTGATGCAG AATTGCAACC AA - #ATGCATGC       1320                                                                          - CTCCTACCTC TTTCAGCAAG ATAAACATTA TGACCTGTCC TATGACACTG GA - #GACAAGGC       1380                                                                          - CTTACAGTGC GGACGCCACG TTGATGTTTT TAAACTATGG CTGATGTGGA GG - #GCAAAGGG       1440                                                                          - GACTACCGGG TTTGAAGCGC ATGTTGATAA ATGTTTGGAG TTGGCAGAGT AT - #TTATACAA       1500                                                                          - CATCATAAAA AACCGAGAAG GATATGAGAT GGTGTTTGAT GGGAAGCCTC AG - #CACACAAA       1560                                                                          - TGTCTGCTTC TGGTACATTC CTCCAAGCTT GCGTACTCTG GAAGACAATG AA - #GAGAGAAT       1620                                                                          - GAGTCGCCTC TCGAAGGTGG CTCCAGTGAT TAAAGCCAGA ATGATGGAGT AT - #GGAACCAC       1680                                                                          - AATGGTCAGC TACCAACCCT TGGGAGACAA GGTCAATTTC TTCCGCATGG TC - #ATCTCAAA       1740                                                                          - CCCAGCGGCA ACTCACCAAG ACATTGACTT CCTGATTGAA GAAATAGAAC GC - #CTTGGACA       1800                                                                          - AGATTTATAA TAACCTTGCT CACCAAGCTG TTCCACTTCT CTAGGTAGAC AA - #TTAAGTTG       1860                                                                          - TCACAAACTG TGTGAATGTA TTTGTAGTTT GTTCCAAAGT AAATCTATTT CT - #ATATTGTG       1920                                                                          - GTGTCAAAGT AGAGTTTAAA AATTAAACAA AAAAGACATT GCTCCTTTTA AA - #AGTCCTTT       1980                                                                          - CTTAAGTTTA GAATACCTCT CTAAGAATTC GTGACAAAAG GCTATGTTCT AA - #TCAATAAG       2040                                                                          - GAAAAGCTTA AAATTGTTAT AAATACTTCC CTTACTTTTA ATATAGTGTG CA - #AAGCAAAC       2100                                                                          - (2) INFORMATION FOR SEQ ID NO:57:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 585 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             #ID NO:57:(xi) SEQUENCE DESCRIPTION: SEQ                                      -      Met Ala Ser Pro Gly Ser Gly Phe - # Trp Ser Phe Gly Ser Glu Asp        Gly                                                                           #   15                                                                        -      Ser Gly Asp Ser Glu Asn Pro Gly - # Thr Ala Arg Ala Trp Cys Gln        Val                                                                           #                 30                                                          -      Ala Gln Lys Phe Thr Gly Gly Ile - # Gly Asn Lys Leu Cys Ala Leu        Leu                                                                           #             45                                                              -      Tyr Gly Asp Ala Glu Lys Pro Ala - # Glu Ser Gly Gly Ser Gln Pro        Pro                                                                           #         60                                                                  -      Arg Ala Ala Ala Arg Lys Ala Ala - # Cys Ala Cys Asp Gln Lys Pro        Cys                                                                           #     80                                                                      -      Ser Cys Ser Lys Val Asp Val Asn - # Tyr Ala Phe Leu His Ala Thr        Asp                                                                           #   95                                                                        -      Leu Leu Pro Ala Cys Asp Gly Glu - # Arg Pro Thr Leu Ala Phe Leu        Gln                                                                           #                110                                                          -      Asp Val Met Asn Ile Leu Leu Gln - # Tyr Val Val Lys Ser Phe Asp        Arg                                                                           #            125                                                              -      Ser Thr Lys Val Ile Asp Phe His - # Tyr Pro Asn Glu Leu Leu Gln        Glu                                                                           #        140                                                                  -      Tyr Asn Trp Glu Leu Ala Asp Gln - # Pro Gln Asn Leu Glu Glu Ile        Leu                                                                           #    160                                                                      -      Met His Cys Gln Thr Thr Leu Lys - # Tyr Ala Ile Lys Thr Gly His        Pro                                                                           #   175                                                                       -      Arg Tyr Phe Asn Gln Leu Ser Thr - # Gly Leu Asp Met Val Gly Leu        Ala                                                                           #                190                                                          -      Ala Asp Trp Leu Thr Ser Thr Ala - # Asn Thr Asn Met Phe Thr Tyr        Glu                                                                           #            205                                                              -      Ile Ala Pro Val Phe Val Leu Leu - # Glu Tyr Val Thr Leu Lys Lys        Met                                                                           #        220                                                                  -      Arg Glu Ile Ile Gly Trp Pro Gly - # Gly Ser Gly Asp Gly Ile Phe        Ser                                                                           #    240                                                                      -      Pro Gly Gly Ala Ile Ser Asn Met - # Tyr Ala Met Met Ile Ala Arg        Phe                                                                           #   255                                                                       -      Lys Met Phe Pro Glu Val Lys Glu - # Lys Gly Met Ala Ala Leu Pro        Arg                                                                           #                270                                                          -      Leu Ile Ala Phe Thr Ser Glu His - # Ser His Phe Ser Leu Lys Lys        Gly                                                                           #            285                                                              -      Ala Ala Ala Leu Gly Ile Gly Thr - # Asp Ser Val Ile Leu Ile Lys        Cys                                                                           #        300                                                                  -      Asp Glu Arg Gly Lys Met Ile Pro - # Ser Asp Leu Glu Arg Arg Ile        Leu                                                                           #    320                                                                      -      Glu Ala Lys Gln Lys Gly Phe Val - # Pro Phe Leu Val Ser Ala Thr        Ala                                                                           #   335                                                                       -      Gly Thr Thr Val Tyr Gly Ala Phe - # Asp Pro Leu Leu Ala Val Ala        Asp                                                                           #                350                                                          -      Ile Cys Lys Lys Tyr Lys Ile Trp - # Met His Val Asp Ala Ala Trp        Gly                                                                           #            365                                                              -      Gly Gly Leu Leu Met Ser Arg Lys - # His Lys Trp Lys Leu Ser Gly        Val                                                                           #        380                                                                  -      Glu Arg Ala Asn Ser Val Thr Trp - # Asn Pro His Lys Met Met Gly        Val                                                                           #    400                                                                      -      Pro Leu Gln Cys Ser Ala Leu Leu - # Val Arg Glu Glu Gly Leu Met        Gln                                                                           #   415                                                                       -      Asn Cys Asn Gln Met His Ala Ser - # Tyr Leu Phe Gln Gln Asp Lys        His                                                                           #                430                                                          -      Tyr Asp Leu Ser Tyr Asp Thr Gly - # Asp Lys Ala Leu Gln Cys Gly        Arg                                                                           #            445                                                              -      His Val Asp Val Phe Lys Leu Trp - # Leu Met Trp Arg Ala Lys Gly        Thr                                                                           #        460                                                                  -      Thr Gly Phe Glu Ala His Val Asp - # Lys Cys Leu Glu Leu Ala Glu        Tyr                                                                           #    480                                                                      -      Leu Tyr Asn Ile Ile Lys Asn Arg - # Glu Gly Tyr Glu Met Val Phe        Asp                                                                           #   495                                                                       -      Gly Lys Pro Gln His Thr Asn Val - # Cys Phe Trp Tyr Ile Pro Pro        Ser                                                                           #                510                                                          -      Leu Arg Thr Leu Glu Asp Asn Glu - # Glu Arg Met Ser Arg Leu Ser        Lys                                                                           #            525                                                              -      Val Ala Pro Val Ile Lys Ala Arg - # Met Met Glu Tyr Gly Thr Thr        Met                                                                           #        540                                                                  -      Val Ser Tyr Gln Pro Leu Gly Asp - # Lys Val Asn Phe Phe Arg Met        Val                                                                           #    560                                                                      -      Ile Ser Asn Pro Ala Ala Thr His - # Gln Asp Ile Asp Phe Leu Ile        Glu                                                                           #   575                                                                       -      Glu Ile Glu Arg Leu Gly Gln Asp - # Leu                                #                585                                                          - (2) INFORMATION FOR SEQ ID NO:58:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 585 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             #ID NO:58:(xi) SEQUENCE DESCRIPTION: SEQ                                      -      Met Ala Ser Pro Gly Ser Gly Phe - # Trp Ser Phe Gly Ser Glu Asp        Gly                                                                           #   15                                                                        -      Ser Gly Asp Pro Glu Asn Pro Gly - # Thr Ala Arg Ala Trp Cys Gln        Val                                                                           #                 30                                                          -      Ala Gln Lys Phe Thr Gly Gly Ile - # Gly Asn Lys Leu Cys Ala Leu        Leu                                                                           #             45                                                              -      Tyr Gly Asp Ser Glu Lys Pro Ala - # Glu Ser Gly Gly Ser Val Thr        Ser                                                                           #         60                                                                  -      Arg Ala Ala Thr Arg Lys Val Ala - # Cys Thr Cys Asp Gln Lys Pro        Cys                                                                           #     80                                                                      -      Ser Cys Pro Lys Gly Asp Val Asn - # Tyr Ala Leu Leu His Ala Thr        Asp                                                                           #   95                                                                        -      Leu Leu Pro Ala Cys Glu Gly Glu - # Arg Pro Thr Leu Ala Phe Leu        Gln                                                                           #                110                                                          -      Asp Val Met Asn Ile Leu Leu Gln - # Tyr Val Val Lys Ser Phe Asp        Arg                                                                           #            125                                                              -      Ser Thr Lys Val Ile Asp Phe His - # Tyr Pro Asn Glu Leu Leu Gln        Glu                                                                           #        140                                                                  -      Tyr Asn Trp Glu Leu Ala Asp Gln - # Pro Gln Asn Leu Glu Glu Ile        Leu                                                                           #    160                                                                      -      Thr His Cys Gln Thr Thr Leu Lys - # Tyr Ala Ile Lys Thr Gly His        Pro                                                                           #   175                                                                       -      Arg Tyr Phe Asn Gln Leu Ser Thr - # Gly Leu Asp Met Val Gly Leu        Ala                                                                           #                190                                                          -      Ala Asp Trp Leu Thr Ser Thr Ala - # Asn Thr Asn Met Phe Thr Tyr        Glu                                                                           #            205                                                              -      Ile Ala Pro Val Phe Val Leu Leu - # Glu Tyr Val Thr Leu Lys Lys        Met                                                                           #        220                                                                  -      Arg Glu Ile Ile Gly Trp Pro Gly - # Gly Ser Gly Asp Gly Ile Phe        Ser                                                                           #    240                                                                      -      Pro Gly Gly Ala Ile Ser Asn Met - # Tyr Ala Met Leu Ile Ala Arg        Tyr                                                                           #   255                                                                       -      Lys Met Phe Pro Glu Val Lys Glu - # Lys Gly Met Ala Ala Val Pro        Arg                                                                           #                270                                                          -      Leu Ile Ala Phe Thr Ser Glu His - # Ser His Phe Ser Leu Lys Lys        Gly                                                                           #            285                                                              -      Ala Ala Ala Leu Gly Ile Gly Thr - # Asp Ser Val Ile Leu Ile Lys        Cys                                                                           #        300                                                                  -      Asp Glu Arg Gly Lys Met Ile Pro - # Ser Asp Leu Glu Arg Arg Ile        Leu                                                                           #    320                                                                      -      Glu Val Lys Gln Lys Gly Phe Val - # Pro Phe Leu Val Ser Ala Thr        Ala                                                                           #   335                                                                       -      Gly Thr Thr Val Tyr Gly Ala Phe - # Asp Pro Leu Leu Ala Val Ala        Asp                                                                           #                350                                                          -      Ile Cys Lys Lys Tyr Lys Ile Trp - # Met His Val Asp Ala Ala Trp        Gly                                                                           #            365                                                              -      Gly Gly Leu Leu Met Ser Arg Lys - # His Lys Trp Lys Leu Asn Gly        Val                                                                           #        380                                                                  -      Glu Arg Ala Asn Ser Val Thr Trp - # Asn Pro His Lys Met Met Gly        Val                                                                           #    400                                                                      -      Pro Leu Gln Cys Ser Ala Leu Leu - # Val Arg Glu Glu Gly Leu Met        Gln                                                                           #   415                                                                       -      Ser Cys Asn Gln Met His Ala Ser - # Tyr Leu Phe Gln Gln Asp Lys        His                                                                           #                430                                                          -      Tyr Asp Leu Ser Tyr Asp Thr Gly - # Asp Lys Ala Leu Gln Cys Gly        Arg                                                                           #            445                                                              -      His Val Asp Val Phe Lys Leu Trp - # Leu Met Trp Arg Ala Lys Gly        Thr                                                                           #        460                                                                  -      Thr Gly Phe Glu Ala His Ile Asp - # Lys Cys Leu Glu Leu Ala Glu        Tyr                                                                           #    480                                                                      -      Leu Tyr Asn Ile Ile Lys Asn Arg - # Glu Gly Tyr Glu Met Val Phe        Asp                                                                           #   495                                                                       -      Gly Lys Pro Gln His Thr Asn Val - # Cys Phe Trp Phe Val Pro Pro        Ser                                                                           #                510                                                          -      Leu Arg Val Leu Glu Asp Asn Glu - # Glu Arg Met Ser Arg Leu Ser        Lys                                                                           #            525                                                              -      Val Ala Pro Val Ile Lys Ala Arg - # Met Met Glu Tyr Gly Thr Thr        Met                                                                           #        540                                                                  -      Val Ser Tyr Gln Pro Leu Gly Asp - # Lys Val Asn Phe Phe Arg Met        Val                                                                           #    560                                                                      -      Ile Ser Asn Pro Ala Ala Thr His - # Gln Asp Ile Asp Phe Leu Ile        Glu                                                                           #   575                                                                       -      Glu Ile Glu Arg Leu Gly Gln Asp - # Leu                                #                585                                                          - (2) INFORMATION FOR SEQ ID NO:59:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 585 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             #ID NO:59:(xi) SEQUENCE DESCRIPTION: SEQ                                      -      Met Ala Ser Pro Gly Ser Gly Phe - # Trp Ser Phe Gly Ser Glu Asp        Gly                                                                           #   15                                                                        -      Ser Gly Asp Ser Glu Asn Pro Gly - # Thr Ala Arg Ala Trp Cys Gln        Val                                                                           #                 30                                                          -      Ala Gln Lys Phe Thr Gly Gly Ile - # Gly Asn Lys Leu Cys Ala Leu        Leu                                                                           #             45                                                              -      Tyr Gly Asp Ala Glu Lys Pro Ala - # Glu Ser Gly Gly Ser Gln Pro        Pro                                                                           #         60                                                                  -      Arg Ala Ala Ala Arg Lys Ala Ala - # Cys Ala Cys Asp Gln Lys Pro        Cys                                                                           #     80                                                                      -      Ser Cys Ser Lys Val Asp Val Asn - # Tyr Ala Phe Leu His Ala Thr        Asp                                                                           #   95                                                                        -      Leu Leu Pro Ala Cys Asp Gly Glu - # Arg Pro Thr Leu Ala Phe Leu        Gln                                                                           #                110                                                          -      Asp Val Met Asn Ile Leu Leu Gln - # Tyr Val Val Lys Ser Phe Asp        Arg                                                                           #            125                                                              -      Ser Thr Lys Val Ile Asp Phe His - # Tyr Pro Asn Glu Leu Leu Gln        Glu                                                                           #        140                                                                  -      Tyr Asn Trp Glu Leu Ala Asp Gln - # Pro Gln Asn Leu Glu Glu Ile        Leu                                                                           #    160                                                                      -      Met His Cys Gln Thr Thr Leu Lys - # Tyr Ala Ile Lys Thr Gly His        Pro                                                                           #   175                                                                       -      Arg Tyr Phe Asn Gln Leu Ser Thr - # Gly Leu Asp Met Val Gly Leu        Ala                                                                           #                190                                                          -      Ala Asp Trp Leu Thr Ser Thr Ala - # Asn Thr Asn Met Phe Thr Tyr        Glu                                                                           #            205                                                              -      Ile Ala Pro Val Phe Val Leu Leu - # Glu Tyr Val Thr Leu Lys Lys        Met                                                                           #        220                                                                  -      Arg Glu Ile Ile Gly Trp Pro Gly - # Gly Ser Gly Asp Gly Ile Phe        Ser                                                                           #    240                                                                      -      Pro Gly Gly Ala Ile Ser Asn Met - # Tyr Ala Met Met Ile Ala Arg        Phe                                                                           #   255                                                                       -      Lys Met Phe Pro Glu Val Lys Glu - # Lys Gly Met Ala Ala Leu Pro        Arg                                                                           #                270                                                          -      Leu Ile Ala Phe Thr Ser Glu His - # Ser His Phe Ser Leu Lys Lys        Gly                                                                           #            285                                                              -      Ala Ala Ala Leu Gly Ile Gly Thr - # Asp Ser Val Ile Leu Ile Lys        Cys                                                                           #        300                                                                  -      Asp Glu Arg Gly Lys Met Ile Pro - # Ser Asp Leu Glu Arg Arg Ile        Leu                                                                           #    320                                                                      -      Glu Ala Lys Gln Lys Gly Phe Val - # Pro Phe Leu Val Ser Ala Thr        Ala                                                                           #   335                                                                       -      Gly Thr Thr Val Tyr Gly Ala Phe - # Asp Pro Leu Leu Ala Val Ala        Asp                                                                           #                350                                                          -      Ile Cys Lys Lys Tyr Lys Ile Trp - # Met His Val Asp Ala Ala Trp        Gly                                                                           #            365                                                              -      Gly Gly Leu Leu Met Ser Arg Lys - # His Lys Trp Lys Leu Ser Gly        Val                                                                           #        380                                                                  -      Glu Arg Ala Asn Ser Val Thr Trp - # Asn Pro His Lys Met Met Gly        Val                                                                           #    400                                                                      -      Pro Leu Gln Cys Ser Ala Leu Leu - # Val Arg Glu Glu Gly Leu Met        Gln                                                                           #   415                                                                       -      Asn Cys Asn Gln Met His Ala Ser - # Tyr Leu Phe Gln Gln Asp Lys        His                                                                           #                430                                                          -      Tyr Asp Leu Ser Tyr Asp Thr Gly - # Asp Lys Ala Leu Gln Cys Gly        Arg                                                                           #            445                                                              -      His Val Asp Val Phe Lys Leu Trp - # Leu Met Trp Arg Ala Lys Gly        Thr                                                                           #        460                                                                  -      Thr Gly Phe Glu Ala His Val Asp - # Lys Cys Leu Glu Leu Ala Glu        Tyr                                                                           #    480                                                                      -      Leu Tyr Asn Ile Ile Lys Asn Arg - # Glu Gly Tyr Glu Met Val Phe        Asp                                                                           #   495                                                                       -      Gly Lys Pro Gln His Thr Asn Val - # Cys Phe Trp Tyr Ile Pro Pro        Ser                                                                           #                510                                                          -      Leu Arg Thr Leu Glu Asp Asn Glu - # Glu Arg Met Ser Arg Leu Ser        Lys                                                                           #            525                                                              -      Val Ala Pro Val Ile Lys Ala Arg - # Met Met Glu Tyr Gly Thr Thr        Met                                                                           #        540                                                                  -      Val Ser Tyr Gln Pro Leu Gly Asp - # Lys Val Asn Phe Phe Arg Met        Val                                                                           #    560                                                                      -      Ile Ser Asn Pro Ala Ala Thr His - # Gln Asp Ile Asp Phe Leu Ile        Glu                                                                           #   575                                                                       -      Glu Ile Glu Arg Leu Gly Gln Asp - # Leu                                #                585                                                          - (2) INFORMATION FOR SEQ ID NO:60:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 18 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             #ID NO:60:(xi) SEQUENCE DESCRIPTION: SEQ                                      -      Gln Lys Phe Thr Gly Gly Ile Gly - # Asn Lys Leu Cys Ala Leu Leu        Tyr                                                                           #   15                                                                        -      Gly Asp                                                                __________________________________________________________________________

We claim:
 1. A monoclonal antibody that binds to a polypeptideconsisting of an amino acid sequence selected from the group consistingof SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 24, SEQ IDNO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 38, SEQID NO: 40, SEQ ID NO: 42, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 48,SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51 and SEQ ID NO:
 60. 2. Themonoclonal antibody of claim 1, wherein said polypeptide consists of SEQID NO:
 17. 3. The monoclonal antibody of claim 1, wherein saidpolypeptide consists of SEQ ID NO:
 19. 4. The monoclonal antibody ofclaim 1, wherein said polypeptide consists of SEQ ID NO:
 21. 5. Themonoclonal antibody of claim 1, wherein said polypeptide consists of SEQID NO:
 24. 6. The monoclonal antibody of claim 1, wherein saidpolypeptide consists of SEQ ID NO:
 32. 7. The monoclonal antibody ofclaim 1, wherein said polypeptide consists of SEQ ID NO:
 33. 8. Themonoclonal antibody of claim 1, wherein said polypeptide consists of SEQID NO:
 34. 9. The monoclonal antibody of claim 1, wherein saidpolypeptide consists of SEQ ID NO:
 35. 10. The monoclonal antibody ofclaim 1, wherein said polypeptide consists of SEQ ID NO:
 38. 11. Themonoclonal antibody of claim 1, wherein said polypeptide consists of SEQID NO:
 40. 12. The monoclonal antibody of claim 1, wherein saidpolypeptide consists of SEQ ID NO:
 42. 13. The monoclonal antibody ofclaim 1, wherein said polypeptide consists of SEQ ID NO:
 45. 14. Themonoclonal antibody of claim 1, wherein said polypeptide consists of SEQID NO:
 47. 15. The monoclonal antibody of claim 1, wherein saidpolypeptide consists of SEQ ID NO:
 48. 16. The monoclonal antibody ofclaim 1, wherein said polypeptide consists of SEQ ID NO:
 49. 17. Themonoclonal antibody of claim 1, wherein said polypeptide consists of SEQID NO:
 50. 18. The monoclonal antibody of claim 1, wherein saidpolypeptide consists of SEQ ID NO:
 51. 19. The monoclonal antibody ofclaim 1, wherein said polypeptide consists of SEQ ID NO:
 60. 20. Ahybridoma cell line capable of producing the monoclonal antibody of anyone of claims 1-18 or 19.